Activation Of Sea Urchin Eggs Research Articles

Central Spindle Self-Organization and Cytokinesis in Artificially Activated Sea Urchin Eggs.

The ability of microtubules of the mitotic apparatus to control the positioning and initiation of the cleavage furrow during cytokinesis was first established from studies on early echinoderm embryos. However, the identity of the microtubule population that imparts cytokinetic signaling is unclear. The two main--and not necessarily mutually exclusive--candidates are the central spindle and the astral rays. In the present study, we examined cytokinesis in ammonia-activated sea urchin eggs, which lack paternally derived centrosomes and undergo mitosis mediated by unusual anastral, bipolar mini-spindles. Live cell imaging and immunolabeling for microtubules and the centralspindlin constituent and kinesin-related protein, MKLP1, demonstrated that furrowing in ammonia-activated eggs was associated with aligned arrays of centralspindlin-linked, opposed bundles of antiparallel microtubules. These autonomous, zipper-like arrays were not associated with a mitotic apparatus, but did possess characteristics similar to the central spindle region of control, fertilized embryos. Our results highlight the self-organizing nature of the central spindle region and its ability to induce cytokinesis-like furrowing, even in the absence of a complete mitotic apparatus.

Rho-kinase in sea urchin eggs and embryos

The activation of sea urchin eggs at fertilization provides an ideal system for studying the molecular events involved in cellular activation. Rho GTPases, which are key signaling enzymes in eukaryotes, are involved in sustaining the activation of sea urchin eggs; however, their downstream effectors have not yet been characterized. In somatic cells, RhoA regulates a serine/threonine kinase known as Rho-kinase (ROCK). The activity of ROCK in early sea urchin development has been inferred, but not tested directly. A ROCK gene was identified in the sea urchin (Strongylocentrotus purpuratus) genome and the sequence of its cDNA determined. The sea urchin ROCK (SpROCK) sequence predicts a protein of 158 kDa with >72% and 45% identities with different protein orthologues of the kinase catalytic domain and the complete protein sequence, respectively. SpROCK mRNA levels are high in unfertilized eggs and decrease to 35% after 15 min postfertilization and remain low up to the 4 cell stage. Antibodies to the human ROCK-I kinase domain revealed SpROCK to be concentrated in the cortex of eggs and early embryos. Co-immunoprecipitation assays indicate that RhoA and SpROCK are physically associated. This association is destroyed by treatment with the C3 exoenzyme and with the ROCK antagonist H-1152. H-1152 also inhibited DNA synthesis in embryos. We conclude that the Rho-dependent signaling pathway, via SpROCK, is essential for early embryonic development.

Bipolar, anastral spindle development in artificially activated sea urchin eggs.

The mitotic apparatus of the early sea urchin embryo is the archetype example of a centrosome-dominated, large aster spindle organized by means of the centriole of the fertilizing sperm. In this study, we tested the hypothesis that artificially activated sea urchin eggs possess the capacity to assemble the anastral, bipolar spindles present in many acentrosomal systems. Control fertilized Lytechinus pictus embryos and ammonia-activated eggs were immunolabeled for tubulin, centrosomal material, the spindle pole structuring protein NuMA and the mitotic kinesins MKLP1/Kinesin-6, Eg5/Kinesin-5, and KinI/Kinesin-13. Confocal imaging showed that a subset of ammonia-activated eggs contained bipolar "mini-spindles" that were anastral; displayed metaphase and anaphase-like stages; labeled for centrosomal material, NuMA, and the three mitotic kinesins; and were observed in living eggs using polarization optics. These results suggest that spindle structural and motor proteins have the ability to organize bipolar, anastral spindles in sea urchin eggs activated in the absence of the paternal centriole.

2DE identification of proteins exhibiting turnover and phosphorylation dynamics during sea urchin egg activation

The animal egg is a unique quiescent cell, prepackaged with maternal mRNAs and proteins that have functions in early development. Rapid, transient signaling at fertilization alters egg physiology, resulting in Ca 2+ release from the endoplasmic reticulum (ER) and cytoplasmic alkalinization. These events trigger the zygote developmental program through initiation of DNA synthesis and entry into mitosis. Post-translational modifications of maternal proteins are responsible for the spatio-temporal regulation that orchestrates egg activation. We used functional proteomics to identify the candidate maternal proteins involved in egg activation and early development. As the first step of this analysis, we present the data on the baseline maternal proteome, in particular, on proteins exhibiting changes in abundance and in phosphorylation state upon egg activation. We identify 94 proteins that were stable, reproducibly displayed a shift in isoelectric point, or changed in relative abundance at specific times after activation. The identities of these proteins were determined by quadrupole time-of-flight tandem mass spectrometry. The set of the most dynamic proteins appear to be enriched in intermediary metabolism proteins, cytoskeletal proteins, gamete associated proteins and proteins that have Ca 2+ mediated activities.

Does cyclic ADP-ribose participate in the activation of sea urchin eggs during fertilization?

Does cyclic ADP-ribose participate in the activation of sea urchin eggs during fertilization?

Detection of centrosome structure in fertilized and artificially activated sea urchin eggs using immunofluorescence microscopy and isolation of centrosomes followed by structural characterization with field emission scanning electron microscopy.

Sea urchin eggs have been used for over a century to study fertilization, cell division, cell differentiation, and embryo development. This system provides excellent and abundant material to investigate the basic mechanisms underlying germ cell functions and to explore tools that facilitate the studies of other less readily available germ cell systems. Immunofluorescence microscopy of microtubule organization performed in sea urchin eggs (,) led to numerous subsequent studies on the cytoskeleton in invertebrate (, , , , , ) and mammalian (, , , ) reproductive cell systems. The pioneering studies on centrosomes in sea urchin eggs were performed over 100 yr ago by Boveri () and have provided a wealth of information and insights that has led to a renaissance and an explosion in centrosome research during the past few years. By using iron hematoxylin as the primary staining technique in fertilized sea urchin eggs, Boveri showed elegantly that dominant centrosome material is contributed by sperm. He showed that sperm centrosomes reorganize after pronuclear fusion and separate to the mitotic poles to form the mitotic apparatus during cell division. Moreover, he showed that sea urchin eggs fertilized with more than one sperm contained supernumerary centrosomes, resulting in multipolar spindles that lead to unequal chromosome separation during cell division. These studies on sea urchin eggs gave thought to the fascinating idea that the formation of multiple centrosome organizations might be involved in the development and progression of malignant tumors ().

A Rho GTPase controls the rate of protein synthesis in the sea urchin egg

Fertilization of the sea urchin egg triggers a Ca 2+-dependent cortical granule exocytosis and cytoskeletal reorganization, both of which are accompanied by an accelerated protein synthesis. The signaling mechanisms leading to these events are not completely understood. The possible role of Rho GTPases in sea urchin egg activation was studied using the Clostridium botulinum C3 exotoxin, which specifically ADP-ribosylates Rho proteins and inactivates them. We observed that incubation of eggs with C3 resulted in in situ ADP-ribosylation of Rho. Following fertilization, C3-treated eggs were capable of performing cortical granule exocytosis but not the first cytokinesis. C3 caused in both unfertilized eggs and early embryos alterations in the state of actin polymerization and inhibition of the spindle formation. Moreover, C3 diminished markedly the rate of protein synthesis. These findings suggested that Rho is involved in regulating the acceleration of protein synthesis that accompanies the egg activation by sperm.

Mastoparan induces Ca 2+-independent cortical granule exocytosis in sea urchin eggs

In most species, cortical granule exocytosis is characteristic of egg activation by sperm. It is a Ca 2+-mediated event which results in elevation of the vitelline coat to block permanently the polyspermy at fertilization. We examined the effect of mastoparan, an activator of G-proteins, on the sea urchin egg activation. Mastoparan was able to induce, in a concentration-dependent manner, the egg cortical granule exocytosis; mastoparan-17, an inactive analogue of mastoparan, had no effect. Mastoparan, but not sperm, induced cortical granule exocytosis in eggs preloaded with BAPTA, a Ca 2+ chelator. In isolated egg cortical lawns, which are vitelline layers and membrane fragments with endogenously docked cortical granules, mastoparan induced cortical granule fusion in a Ca 2+-independent manner. By contrast, mastoparan-17 did not trigger fusion. We conclude that in sea urchin eggs mastoparan stimulates exocytosis at a Ca 2+-independent late site of the signaling pathway that culminates in cortical granule discharge.

Probable participation of phospholipase A2 reaction in the process of fertilization-induced activation of sea urchin eggs.

In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a phospholipase A2 inhibitor) blocked fertilization envelope formation and transient CN(-)-insensitive respiration in a concentration-dependent manner. BPB had virtually no effect on the increase in [Ca2+]i (cytosolic Ca2+ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN(-)-sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN(-)-insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A2, induced the envelope formation, acid production, augmentation of CN(-)-insensitive and sensitive respiration, but did not cause any increase in [Ca2+]i, the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A2 induced by Ca2+ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN(-)-insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca(2+)-triggered sequential reactions other than Ca(2+)-caused activation of phospholipase A2.

Internal calcium release and activation of sea urchin eggs by cGMP are independent of the phosphoinositide signaling pathway.

We show that microinjecting cyclic GMP (cGMP) into unfertilized sea urchin eggs activates them by stimulating a rise in the intracellular free calcium ion concentration ([Ca2+]i). The increase in [Ca2+]i is similar in both magnitude and duration to the transient that activates the egg at fertilization. It is due to mobilization of calcium from intracellular stores but is not prevented by the inositol trisphosphate (InsP3) antagonist heparin. Furthermore, cGMP does not stimulate the eggs Na+/H+ antiport when the [Ca2+]i transient is blocked by the calcium chelator bis-(O-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), suggesting that cGMP does not activate eggs by interacting with the their phosphoinositide signaling pathway. However, the [Ca2+]i increase and activation are prevented in eggs in which the InsP3-sensitive calcium stores have been emptied by the prior microinjection of the InsP3 analogue inositol 1,4,5-trisphosphorothioate. These data indicate that cGMP activates eggs by stimulating the release of calcium from an InsP3-sensitive calcium store via a novel, though unidentified, route independent of the InsP3 receptor.

Effects of quinacrine on egg activation: A possible role for phospholipase A2in sea urchin fertilization

Summary The possible role of arachidonic acid and its metabolites in sea urchin egg activation was examined using inhibitors of arachidonic acid release and metabolism. Quinacrine, a phospholipase A2 inhibitor, blocked the cortical granule reaction and inhibited pronuclear migration and cleavage in fertilized eggs. Indomethacin and eicosatetrayenoic acid, blockers of arachidonic acid metabolism, had no affect on these processes. These findings suggest that phospholipase A2 activation may be important in mediating events involving cortical granule exocytosis, pronuclear morphogenesis and mitosis in fertilized sea urchin eggs, perhaps by releasing free arachidonic acid. Subsequent metabolism of arachidonic acid does not seem to be involved in processes of egg activation as inhibitors of arachidonic acid metabolism had no demonstrable effects on fertilization events following egg activation and the cortical granule reaction.

Tyrosine kinase activity at fertilization: Possible role of phospholipase C-gamma at activation of sea urchin egg

Tyrosine kinase activity at fertilization: Possible role of phospholipase C-gamma at activation of sea urchin egg

A specific requirement for protein kinase C in activation of the respiratory burst oxidase of fertilization.

Partially reduced oxygen species are toxic, yet activated sea urchin eggs produce H2O2, suggesting that the control of oxidant stress might be critical for early embryonic development. We show that the Ca2(+)-stimulated NADPH oxidase that generates H2O2 in the "respiratory burst" of fertilization is activated by a protein kinase, apparently to regulate the synthesis of this potentially lethal oxidant. The NADPH oxidase was separated into membrane and soluble fractions that were both required for H2O2 synthesis. The soluble fraction was further purified by anion exchange chromatography. The factor in the soluble fraction that activated the membrane-associated oxidase was demonstrated to be protein kinase C (PKC) by several criteria, including its Ca2+/phophatidylserine/diacyl-glycerol-stimulated histone kinase activity, its response to phorbol ester, its inhibition by a PKC pseudosubstrate peptide, and its replacement by purified mammalian PKC. Neither calmodulin-dependent kinase II, the catalytic subunit of cyclic AMP-dependent protein kinase, casein kinase II, nor myosin light chain kinase activated the oxidase. Although the PKC family has been ubiquitously implicated in cellular regulation, enzymes that require PKC for activation have not been identified; the respiratory burst oxidase is one such enzyme.

Relationships between intracellular pH, protein synthesis, and actin assembly during parthenogenetic activation of sea urchin eggs

The relationship between the rate of protein synthesis and the state of assembly of actin was investigated in parthenogenetically activated and fertilized green sea urchin (Strongylocentrotus droebachiensis) eggs. Artificial agents known to raise the intracellular pH (pHi) without affecting the internal calcium concentration were used. Incubation of the eggs with ammonia, procaine, or 12-O-tetradecanoylphorbol 13-acetate (TPA, a protein kinase C activator) resulted in an increase of the protein synthesis rate. At low amounts of ammonia and procaine, this increase was concentration dependent. Higher concentrations of these amine compounds were inhibitory to protein synthesis. Also, chromosome condensation was optimal only in the eggs treated with low concentrations of ammonia or procaine, and was never observed in the TPA-treated eggs. None of these treatments induced detectable changes of the initial amount of G-actin observed in the resting cell, as measured by the deoxyribonuclease I assay. In parallel, no changes of the F-actin content were detected, as determined by quantitative fluorometric measurements of rhodamine–phalloidin labeled eggs. On the other hand, fertilized eggs that showed a higher rate of protein synthesis than parthenogenetically activated eggs were also characterized by a decrease in the total cellular concentration of G-actin. Thus, whereas protein synthesis and chromosome condensation may proceed in the absence of any polymerization of actin in the parthenogenetically activated eggs, it cannot be excluded that actin assembly could be linked to this metabolic activity in the fertilized egg.

Activation of sea urchin eggs by inositol phosphates is independent of external calcium.

We investigated the contribution of external calcium ions to inositol phosphate-induced exocytosis in sea urchin eggs. We show that: (a) inositol phosphates activate eggs of the sea urchin species Lytechinus pictus and Lytechinus variegatus independently of external calcium ions; (b) the magnitude and duration of the inositol phosphate induced calcium changes are independent of external calcium; (c) in calcium-free seawater, increasing the volume of inositol trisphosphate solution injected decreased the extent of egg activation; (d) eggs in calcium-free sea water are more easily damaged by microinjection; microinjection of larger volumes increased leakage from eggs pre-loaded with fluorescent dye. We conclude that inositol phosphates do not require external calcium ions to activate sea urchin eggs. This is entirely consistent with their role as internal messengers at fertilization. The increased damage caused to eggs in calcium-free seawater injected with large volumes may allow the EGTA present in the seawater to enter the egg and chelate any calcium released by the inositol phosphates. This may explain the discrepancy between this and earlier reports.

Activation of Sea Urchin Eggs by Halothane and Its Inhibition by Dantrolene: (sea urchin eggs/fertilization/halothane/dantrolene/nifedipine).

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. 45 Ca2+ influx was substantially stimulated by halothane in eggs exposed to 45 CaCl2 . Halothane-induced 45 Ca2+ 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.

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.

Kinetics of actin assembly attending fertilization or artificial activation of sea urchin eggs

Changes in the state of actin assembly triggered by fertilization or by artificial activation of sea urchin eggs were quantified using the DNase I inhibition assay. Insemination of Lytechinus pictus or Strongylocentrotus purpuratus eggs induces a cyclic variation in the level of G-actin as follows: between 0 and 30 s after insemination, the G-actin content decreases. This is followed by an increase in the amount of monomeric actin between 30 and 60 s, and then from 60 s to 5 min postinsemination there is a progressive decrease in the egg's level of G-actin. This latter decrease is more pronounced in S. purpuratus eggs than in L. pictus eggs. Using sperm mimetics that trigger an increase in intracellular calcium concentration (A23187 in sodium-free seawater), a cytoplasmic alkalinization (NH 4Cl), a plasma membrane depolarization (seawater enriched with potassium ions), or all three of these phenomena (A23187 in normal seawater), each phase depicted at fertilization correlates with the following metabolic events accompanying egg awakening: phase 1, of uncertain origin (possibly related to plasma membrane depolarization); phase 2, elevation of intracellular calcium concentration; phase 3, alkalinization of the intracellular milieu but only if the transient intracellular calcium rise has taken place.

Inositol(1,3,4,5)tetrakisphosphate-induced activation of sea urchin eggs requires the presence of inositol trisphosphate

We have earlier reported that Inositol(1,3,4,5)tetrakisphosphate microinjection will activate eggs of the sea urchin Lytechinus variegatus provided that it is co-injected with inositol(2,4,5)trisphosphate (Irvine and Moor, Biochem. J. 240 , 917–920, 1986). Here we extend these observations to show that (1) inositol(1,3,4,5,6)pentakisphosphate is a partial agonist in this assay and (2) the requirement for the presence of inositol(2,4,5)trisphosphate cannot be bypassed by raised, but sub-threshold, Ca 2+ concentrations. A mechanism for the proposed stimulation of Ca 2+ entry into the cell requiring both inositol tris- and tetrakisphosphates is presented.

The part played by inositol trisphosphate and calcium in the propagation of the fertilization wave in sea urchin eggs.

Sea urchin egg activation at fertilization is progressive, beginning at the point of sperm entry and moving across the egg with a velocity of 5 microns/s. This activation wave (Kacser, H., 1955, J. Exp. Biol., 32:451-467) has been suggested to be the result of a progressive release of calcium from a store within the egg cytoplasm (Jaffe, L. F., 1983, Dev. Biol., 99:265-276). The progressive release of calcium may be due to the production of inositol trisphosphate (InsP3), a second messenger. We show here that a wave of calcium release crosses the Lytechinus pictus egg; the peak of the wave travels with a velocity of 5 microns/s; microinjection of InsP3 causes the release of calcium within the egg; calcium release (as judged by fertilization envelope elevation) is abolished by prior injection of the calcium chelator EGTA; neomycin, an inhibitor of InsP3 production, does not prevent the release of calcium in response to InsP3 but does abolish the wave of calcium release; the egg cytoplasm rapidly buffers microinjected calcium; the calcium concentration required to cause fertilization membrane elevation when microinjected is very similar to that required to stimulate the production of InsP3 in vitro; and the progressive fertilization membrane elevation seen after microinjection of calcium buffers appears to be due to diffusion of the buffer across the egg cytoplasm rather than to the induction of the activation wave. We conclude that InsP3 diffuses through the egg cytoplasm much more readily than calcium ions and that calcium-stimulated production of InsP3 and InsP3-induced calcium release from an internal store can account for the progressive release of calcium at fertilization.