Alteration of sea urchin embryo cell surface properties by mycostatin, a sterol binding antibiotic

Sea urchin embryos incubated in sea water containing mycostatin (MST), a polyene antibiotic, dissociate into single cells. Reaggregation of dissociated sea urchin embryo cells, and uptake of labeled precursors by these cells are also greatly inhibited although O₂ consumption is only slightly affected by this compound. It is known that mycostatin binds primarily to membrane sterols and affects only cells containing membrane sterols. Sea urchin cell membranes contain sterols. The effects of mycostatin on cell adhesion, reaggregation, and permeability seen in this study may be a result of an interaction with cell membrane sterols or sterol-associated molecules.     

Rho, Rho-kinase, and the actin cytoskeleton regulate the Na+ -H+ exchanger in sea urchin eggs

At fertilization, the sea urchin egg undergoes an internal pH (pHi) increase mediated by a Na+ -H+ exchanger. We used antibodies against the mammalian antiporters NHE1 and NHE3 to characterize this exchanger. In unfertilized eggs, only anti-NHE3 cross-reacted specifically with a protein of 81-kDa, which localized to the plasma membrane and cortical granules. Cytochalasin D, C3 exotoxin (blocker of RhoGTPase function), and Y-27632 (inhibitor of Rho-kinase) prevented the pHi change in fertilized eggs. These inhibitors blocked the first cleavage division of the embryo, but not the cortical granule exocytosis. Thus, the sea urchin egg has an epithelial NHE3-like Na+ -H+ exchanger which can be responsible for the pHi change at fertilization. Determinants of this pHi change can be: (i) the increase of exchangers in the plasma membrane (via cortical granule exocytosis) and (ii) Rho, Rho-kinase, and optimal organization of the actin cytoskeleton as regulators, among others, of the intrinsic activity of the exchanger.     

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.     

Identification and characterization of PlAlix,the Alix homologue from the Mediterranean sea urchin Paracentrotus lividus

The sea urchin provides a relatively simple and tractable system for analyzing the early stages of embryo development. Here, we use the sea urchin species, Paracentrotus lividus, to investigate the role of Alix in key stages of embryogenesis, namely the egg fertilization and the first cleavage division. Alix is a multifunctional protein involved in different cellular processes including endocytic membrane trafficking, filamentous (F)‐actin remodeling, and cytokinesis. Alix homologues have been identified in different metazoans; in these organisms, Alix is involved in oogenesis and in determination/differentiation events during embryo development. Herein, we describe the identification of the sea urchin homologue of Alix, PlAlix. The deduced amino acid sequence shows that Alix is highly conserved in sea urchins. Accordingly, we detect the PlAlix protein cross‐reacting with monoclonal Alix antibodies in extracts from P. lividus, at different developmental stages. Focusing on the role of PlAlix during early embryogenesis we found that PlAlix is a maternal protein that is expressed at increasingly higher levels from fertilization to the 2‐cell stage embryo. In sea urchin eggs, PlAlix localizes throughout the cytoplasm with a punctuated pattern and, soon after fertilization, accumulates in larger puncta in the cytosol, and in microvilli‐like protrusions. Together our data show that PlAlix is structurally conserved from sea urchin to mammals and may open new lines of inquiry into the role of Alix during the early stages of embryo development.     

Localization and possible function of 20 kDa actin-modulating protein (actolinkin) in the sea urchin egg

We have previously described a novel actin-capping protein, a 20,000-molecular weight protein (20K protein)-actin complex (20K-A) isolated from sea urchin eggs. In the present study, the localization and possible function of this 20K protein were investigated. The 20K protein was localized in the sea urchin egg cortex. Its distribution in the cortex as revealed by immunofluorescence microscopy did not change during or after fertilization up to the first mitosis, but it was concentrated to some extent in the cleavage furrow region. Exogenously added actin polymerized on the cortex isolated from unfertilized egg; however, actin did not polymerize on the cortex extracted with 0.6 M KCl, that is, the cell membrane, which lost the 20K protein. The cell membrane preincubated with 20K-A restored the activity to grow actin filaments. When decorated with myosin subfragment 1, almost all the actin filaments showed the arrowhead configuration pointing away from the membrane, indicating that they were connected to the membrane at their barbed ends. These results strongly suggest that the 20K protein connects actin filaments to the plasma membrane of sea urchin eggs. Because of this property we call this protein "actolinkin".     

Inhibition of mitosis in fertilized sea urchin eggs by inhibition of the cyclic AMP-dependent protein kinase

Inhibition of cAMP-dependent protein kinase activity by microinjection of a specific physiologic protein inhibitor into sea urchin eggs inhibits the first cleavage after fertilization. Inhibition apparently occurs at some time prior to or during formation of the mitotic spindle. Measurement of the total protein kinase activity of sea urchin egg homogenates after fertilization showed that cAMP-dependent phosphorylation increases after fertilization and then declines prior to or at the time of the first cleavage. It is concluded that a cAMP-dependent phosphorylation plays a significant role in events leading to regulation of mitotic spindle assembly.     

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.     

K+ activity and regulation of intracellular pH in the sea urchin egg during fertilization

Fertilization of the sea urchin egg is accompanied by changes in intracellular ion activities and transmembrane fluxes, which regulate the sequence of biochemical events of metabolic derepression. Changes in intracellular K+ activity during fertilization have been controversial and here we report our measurements using intracellular K+-sensitive microelectrodes. A small, but statistically significant, transient rise in internal K+ activity was detected during the first 10 min of fertilization. Since this change in K+ activity was ouabain sensitive, intracellular K+ activity in the fertilized egg appears to be regulated by the increased Na+, K+ ATPase activity, rather than the previously suggested K+ decompartmentalization. Increasing external K+ concentration was found to stimulate ouabain-sensitive alkalinization in the fertilized egg. The data are consistent with the possibility that Na+, K+ ATPase may regulate cytoplasmic pH by recycling Na+ that enters the cell through Na+-H+ antiport.     

Effect of various ionic compositions upon the membrane potentials during activation of sea urchin eggs

The membrane potentials of sea urchin (Hemicentrotus pulcherrimus) eggs before and after fertilization and their changes during the membrane elevation induced by intracellular electrical stimulation were recorded in solutions of various ionic compositions. Upon fertilization, the membrane potential (?10 mV) depolarized and reversed polarity by a few mV, then gradually returned to a new steady level ranging between ?50 and ?60 mV. The activation potential is closely associated with a transient increase in the membrane permeability. The potential of the unfertilized egg is hyperpolarized by monovalent anions (Br^?, Cl^? and NO₃^?) and depolarized slightly by K⁺. In contrast, the membrane of the fertilized egg is markedly depolarized by K⁺. Suppression of depolarization associated with an increase of the membrane permeability was recorded in Na-free medium (Tris-HCl). The selective increase in permeability to monovalent anions is thought to alternate with the selective increase in permeability to K⁺through the mediation of a transient increase of Na⁺-permeability at the time of fertilization. No causal relationship between the membrane elevation and the depolarization was established because the breakdown of the cortical granules occurs without depolarization or an increase in membrane permeability.     

Phosphorylation-dependent regulation of skeletogenesis in sea urchin micromere-derived cells and embryos

Sea urchin embryo micromeres when isolated and cultured in vitro differentiate to produce spicules. Although several authors have used this model, almost nothing is known about the signaling pathways responsible for initiating skeletogenesis. In order to investigate the potential involvement of phosphorylation events in spiculogenesis, the effect of inhibitors of protein kinases and phosphatases on skeleton formation was studied. Results obtained using both cultured micromeres and embryos revealed that protein tyrosine kinase and phosphatase inhibitors blocked skeleton formation, but not serine/threonine phosphatase inhibitors. The inhibitors showed a dose-dependent effect and when removed from micromere or embryo culture, spicule formation resumed. Inhibition of tyrosine phosphatases resulted in an increase in the tyrosine phosphorylation level of two major proteins and a modest decrease in the expression of the mRNA coding for type I fibrillar collagen. These findings strongly suggest that tyrosine phosphorylation and dephosphorylation is required for micromere differentiation and for normal skeletogenesis during sea urchin embryo development.     

Chloride permeability of sea urchin eggs.

We have examined the content and permeability of chloride in sea urchin eggs. After fertilization there is a large increase in the permeability to chloride. We discuss the mechanism underlying this permeability change and the generalized increase in ion permeability observed after 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.     

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.     

A protein of the basal lamina of the sea urchin embryo

The purification, biochemical characterization and functional features of a novel extracellular matrix protein are described. This protein is a component of the basal lamina found in embryos from the sea urchin species Paracentrotus lividus and Hemicentrotus pulcherrimus . The protein has been named PI-200 K or Hp-200 K, respectively, because of the species from which it was isolated and its apparent molecular weight in SDS-PAGE under reducing conditions. It has been purified from unfertilized eggs where it is found packed within cytoplasmic granules, and has different binding affinities to type I collagen and heparin, as assessed by affinity chromatography columns. By indirect immunofluorescence experiments it was shown that, upon fertilization, the protein becomes extracellular, polarized at the basal surface of ectoderm cells, and on the surface of primary mesenchyme cells at the blastula and gastrula stages. The protein serves as an adhesive substrate, as shown by an in vitro binding assay where cells dissociated from blastula embryos were settled on 200K protein-coated substrates. To examine the involvement of the protein in morphogenesis of sea urchin embryo, early blastula embryos were microinjected with anti-200K Fab fragments and further development was followed. When control embryos reached the pluteus stage, microinjected embryos showed severe abnormalities in arms and skeleton elongation and patterning. On the basis of current results, it was proposed that 200K protein is involved in the regulation of sea urchin embryo skeletogenesis.     

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.     

Intracellular sodium activity in the sea urchin egg during fertilization

Fertilization of the sea urchin egg triggers a sequence of events that are necessary for metabolic derepression and stimulation of proliferation. Changes in intracellular Ca2+ and H+ activities regulate the sequence of events. Intracellular sodium activity is important in the regulation of the intracellular activities of these ions and may directly regulate metabolic events. Using Na+-sensitive microelectrodes we continuously measured the intracellular Na+ activity during fertilization. The results show an increase in intracellular sodium activity medicated by two pathways of Na+ entry: Na+ permeability increase during the fertilization potential and initiation of Na+-H+ exchange activity. Intracellular Na+ activity returned to unfertilized levels by 20 min after fertilization. This decrease was inhibited by ouabain, which suggests the activation of Na+, K+ ATPase during fertilization.     

Identification of GTP-Binding Proteins by ADP-Ribosylation in the Presence of Cholera Toxin, Pertussis Toxin and Botulinum Toxin D in Plasma Membrane Isolated from Eggs and Embryos of Sea Urchin

In plasma membrane fraction isolated from eggs and embryos of sea urchin, ³²P-labeled proteins were found on the fluorographs of SDS-polyacrylamide gel electrophoresis, performed after an exposure of the fraction to [adenylate-³²P] nicotinamide adenine dinucleotide in the presence of cholera toxin, pertussis toxin or botulinum toxin D. The molecular weights of proteins, thus ADP-ribosylated in the presence of cholera toxin and pertussis toxin are 45 and 39 K, which correspond to Gs and Gi or Go, respectively. Protein with the molecular weight of 24 K, labeled in the presence of botulinum toxin D, corresponds to small molecular weight G-protein. The labeling intensity of 45 K protein, probably proportional to its amount, became high at the blastula stage. The labeling intensity of 39 K protein was hardly altered up to the blastula stage. The labeling intensity of 24 K protein increased after fertilization and further increase occurred at the blastula stage. At the gastrula stage, the labeling intensities of these proteins became somewhat lower than at the blastula stage. Transmembrane signaling system, in which these G-proteins are involved, is probably altered in its function during early development.     

The Effects of Chemical Animalizing and Vegetalizing Agents and of Cell Dissociation on the Synthesis of 5S RNA and Transfer RNA in Cleaving Sea Urchin Embryos

The effect of altering normal cell associations and interactions on the synthesis of 5S RNA and transfer RNA (tRNA) was studied in cleaving embryos of the sea urchin, Arbacia punctulata. Cell interactions were altered: (1) by culturing cleaving embryos in the animalizing agent, Evans Blue, and in the vegetalizing agent, Li⁺ as LiCl and (2) by culturing dissociated cells. Control and experimental embryos each were labeled from 3 h to 6 h post fertilization with [8-³H]-guanosine. Sixteen-cell embryos, whose GTP precursor pools had been preloaded, were dissociated, labeled and cultured under conditions which prevent reaggregation. Quantitative measurements of rates of accumulation of newly synthesized 5S RNA and tRNA showed that these rates are similar in cleaving sea urchin embryos and in corresponding embryos cultured in the presence of Evans Blue and of Li⁺. In addition, cells dissociated from cleavage embryos and maintained under conditions which prevent reaggregation retained the ability to synthesize 5S RNA and tRNA. These results suggest that normal cell associations and interactions are not necessary for the synthesis of 5S RNA and tRNA to occur in cleaving sea urchin embryos.     

Real-time and noninvasive monitoring of respiration activity of fertilized ova using semiconductor-based biosensing devices

In this report, we propose a novel evaluation method of embryo activity, describing the real-time and noninvasive electrical monitoring of embryo activity, caused by fertilization of the sea urchin, using a biologically-coupled field-effect transistor (bio-FET) comprised of semiconductor-based biosensing devices. The detection principle of bio-FET is based on the potentiometric detection of charge density change at the gate insulator, which includes changes of hydrogen ion concentration corresponding to pH variation. The surface potential at the gate surface of the bio-FET increased after the introduction of sperms into the ova, resulting in fertilization on the gate sensing area. The positive shift of surface potential indicates the increase of positive charges of hydrogen ions generated by dissolved carbon dioxide in artificial sea water based on respiration activity of the embryo. Moreover, the electrical signal of embryo activity is suppressed due to the inhibition of cytokinesis by introduction of cytochalasin B. The platform based on the bio-FET is expected to be a real-time, label-free and noninvasive detection system, not only in fundamental studies of embryo activity but also in the evaluation of embryo quality for in vitro fertilization.