Carbon dioxide efflux accompanies release of fertilization acid from sea urchin eggs.

Cytoplasmic tubulin purified from unfertilized sea urchin eggs self-assembles in the absence of microtubule-associated proteins (MAPs) [Suprenant and Rebhun, 1983; Detrich and Wilson, 1983] with a critical concentration for polymerization of 0.8 mg/ml at 15-18 degrees C, a value well below the 3 mg/ml tubulin present in these eggs [Pfeffer et al, 1976]. Studies of the calcium sensitivity of unfertilized S. purpuratus (sea urchin) egg tubulin were initiated to help understand how this tubulin is maintained unassembled in the unfertilized egg. Egg microtubules, assembled at physiological temperatures (15-18 degrees C) were depolymerized by a 100-fold lower free calcium concentration than egg microtubules assembled at the higher temperatures (25-37 degrees C) generally used to assemble mammalian brain microtubules. The initial rate of egg microtubule assembly was much more sensitive to calcium than was microtubule depolymerization at steady state at 37 degrees C. However, both processes were sensitive to near physiological free calcium concentrations at 18 degrees C. The co-assembly of bovine brain MAPs and sea urchin egg tubulin produced microtubules that required a 1,000-fold higher concentration of free calcium for depolymerization than microtubules assembled at 18 degrees C from egg tubulin alone. While calcium regulatory MAPs have not yet been found in sea urchin eggs, the fact that brain MAPs interact with egg tubulin and regulate both its critical concentration for polymerization [Suprenant and Rebhun, 1983] and its calcium sensitivity, suggests that such regulatory molecules exist. These results suggest that sea urchin egg tubulin assembly in vivo could be controlled by variations in intracellular calcium levels acting in concert with urchin egg proteins similar in function to brain MAPs.

Translational initiation factors from sea urchin eggs and embryos: Functional properties are highly conserved

Unfertilized eggs of animals contain maternal messenger RNAs (mRNAs) and proteins, which are required for the maintenance of metabolism and regulation of development during the initial stages of embryogenesis. Unfertilized eggs are transcriptionally and translationally quiescent. After fertilization, activated translation of maternal mRNAs is one of the major forces that direct the early stages of embryogenesis before activation of the zygotic genome. However, a low rate and level of protein synthesis have been detected in unfertilized sea urchin eggs indicating that translation is not completely inhibited. Analysis of translatomes of unfertilized eggs and early embryos detected three sets of maternal mRNAs translated either before or after fertilization, or both before and after fertilization. Proteins encoded by maternal mRNAs, which are translated in unfertilized eggs, perform many different functions required for homeostasis, fertilization, egg activation, and early development. This suggests that translation in unfertilized sea urchin eggs may be required to renew the pool of proteins involved in these processes. Thus, translation may be necessary to maintain the fertility and developmental potential of sea urchin eggs during the long-term storage of eggs in ovaries until spawning begins.

EmbryologiaVolume 9, Issue 1 p. 34-39 Free Access THE SODIUM AND POTASSIUM CONTENT IN UNFERTILIZED AND FERTILIZED EGGS OF THE SEA URCHIN REIJI HORI, REIJI HORI Biological Institute, Toyama University, Toyama This work was partly supported by the Scientific Fund of the Ministry of Education.Search for more papers by this author REIJI HORI, REIJI HORI Biological Institute, Toyama University, Toyama This work was partly supported by the Scientific Fund of the Ministry of Education.Search for more papers by this author First published: October 1965 https://doi.org/10.1111/j.1440-169X.1965.tb00213.xCitations: 13 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Literature Biakaszewicz, K., 1929. Recherches sur la réporition des electrolytes dans le protoplasme des cellules ovalarires. Protoplasm, 6, 1– 50. Brooks, S. C., 1940. The intake of radioactive isotopes by living cells. Cold Spring Harbor Symp. Quant. Biol., 8, 171– 180. Hiramoto, Y., 1959a. Changes in electric properties upon fertilization in the sea urchin egg. Exptl. Cell Res., 16, 421– 424. Hiramoto, Y. 1959b. Electric properties of echinoderm eggs. Embryologia, 4, 219– 235. Hodgkin, A. L., 1951. The ionic basis of electrical activity in nerve and muscle. Biol. Rev., 26, 339– 409. Hori, R., 1957. Membrane potential of the unfertilized egg of the Medaka, Oryzias latipes and its changes accompanying activation (in Japanese). Zool. Mag., 66, 87. Hori, R. 1958. On the change in membrane potential accompanying fertilization of the narcotized egg of the sea urchin, Hemicentrotus pulcherrimus. Bull. Marine Biol. Stat. Asamushi, 9, 67– 68. Lord Rothschild and H. Barnes, 1953. The inorganic constituents of the sea urchin egg. J. exp. Biol., 30, 534– 544. Malm, M. and L. Wachtmeister, 1950. A comparison between the potassium content of unfertilized and fertilized sea-urchin eggs. Arkiv för Kemi., 2, 443– 449. Monroy-Oddo, A. and M. Esposito, 1951. Changes in the potassium content of sea urchin eggs on fertilization. J. gen. Physiol., 34, 285– 293. Nakamura, M., 1950. Phosphate determination in biological materials (in Japanese). Agric. Chem., 24, 1– 8. Okuno, H., M. Honda, and H. Ishimori, 1953. Separation of alkali metals with ion-exchange resine (in Japanese). Japan Analyst, 2, 428– 431. Page, I. H., 1927. The electrolyte content of the sea urchin and starfish egg. Biol. Bull., 52, 168– 172. Scheer, B. T., A. Monroy, M. Santangelo, and G. Riccobono, 1954. Action potentials in sea urchin egg at fertilization. Exptl. Cell Res., 7, 284– 287. Shapiro, H. and H. Davson, 1941. Permeability of the Arbacia egg to potassium. Biol. Bull., 81, 295– 296. Sugiyama, M., 1947. On the artificial parthenogenesis of sea urchin egg II. Membrane formation with urea solution and salt ions (in Japanese). Zool. Mag., 57, 79– 80. Tyler, A. and A. Monroy, 1959. Changes in the rate of transfer of potassium across the membrane upon fertization of eggs of Arbacia punctulata. J. exp. Zool., 142, 675– 690. Tyler, A. and A. Monroy, C. Y. Kao, and H. Grundfest, 1956. Membrane potential and resistance of the starfish egg before and after fertilization. Biol. Bull, 111, 153– 177. Citing Literature Volume9, Issue1October 1965Pages 34-39 ReferencesRelatedInformation

The effect of D2O on the crystallization of polymerizable tubulin in sea urchin egg cytoplasm was investigated by estimating the yield of "vinblastine (VB)-crystals" by directly measuring the dimensions of the crystals produced and by protein assays of the crystal isolates. The yield of VB-crystals in mature unfertilized eggs was fairly constant; it neither increased nor decreased in the presence of D2O. On fertilization, the yield of crystals decreased markedly as compared with yields from unfertilized eggs; but, the yield was restored to the value for unfertilized eggs when an adequate concentration of D2O was present during incubation. These results are evidence that tubulin molecules in unfertilized sea urchin eggs are in the polymerizable state but become masked and partly unpolymerizable after fertilization and the D2O releases the masked state and converts unpolymerizable tubulin molecules into active and polymerizable state.

The in Vivo Rate of Glucose-6-Phosphate Dehydrogenase Activity in Sea Urchin Eggs Determined with a Photolabile Caged Substrate

The cortical actin cytoskeleton undergoes dramatic rearrangements during fertilization of sea urchin eggs. To characterize these changes further, we quantified the relative changes in filamentous actin (F-actin) during fertilization and the first cell cycle in both intact eggs and in isolated cortices by quantitative fluorescence microscopy. The level of F-actin in the intact egg decreased after fertilization and continued to decrease throughout the first cell cycle. By 60 min after fertilization, the level of F-actin had decreased to 50% of the unfertilized sea urchin egg. By cytokinesis, the level of F-actin had decreased to 30% of the unfertilized egg. After completion of cell division, individual blastomeres had 10% of the F-actin in the unfertilized egg. In contrast, there was an increase in cortical F-actin to 370% of the level in the unfertilized egg after fertilization. This increase corresponded to the formation of microvilli. There was little change in the level of cortical F-actin during the first cell cycle. We draw parallels to other systems that increase the amount of F-actin in the Triton-insoluble cytoskeleton by recruiting actin from a Triton-soluble pool of F-actin. © 1996 Wiley-Liss, Inc.

The cortical actin cytoskeleton undergoes dramatic rearrangements during fertilization of sea urchin eggs. To characterize these changes further, we quantified the relative changes in filamentous actin (F-actin) during fertilization and the first cell cycle in both intact eggs and in isolated cortices by quantitative fluorescence microscopy. The level of F-actin in the intact egg decreased after fertilization and continued to decrease throughout the first cell cycle. By 60 min after fertilization, the level of F-actin had decreased to 50% of the unfertilized sea urchin egg. By cytokinesis, the level of F-actin had decreased to 30% of the unfertilized egg. After completion of cell division, individual blastomeres had 10% of the F-actin in the unfertilized egg. In contrast, there was an increase in cortical F-actin to 370% of the level in the unfertilized egg after fertilization. This increase corresponded to the formation of microvilli. There was little change in the level of cortical F-actin during the first cell cycle. We draw parallels to other systems that increase the amount of F-actin in the Triton-insoluble cytoskeleton by recruiting actin from a Triton-soluble pool of F-actin.

Previous articleNext article No AccessConference on Problems of General and Cellular Physiology Relating to Fertilization. IIIIon Exchanges and Fertilization in Echinoderm EggsEdward L. Chambers and Robert ChambersEdward L. Chambers Search for more articles by this author and Robert Chambers Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The American Naturalist Volume 83, Number 813Nov. - Dec., 1949 Published for The American Society of Naturalists Article DOIhttps://doi.org/10.1086/281605 Views: 3Total views on this site Citations: 20Citations are reported from Crossref PDF download Crossref reports the following articles citing this article:John D. Biggers, R. Michael Borland, R. Douglas Powers Transport Mechanisms in the Preimplantation Mammalian Embryo, (May 2008): 129–153.https://doi.org/10.1002/9780470720332.ch7Sheldon S. Shen, An-Li Sui K+ activity and regulation of intracellular pH in the sea urchin egg during fertilization, Experimental Cell Research 183, no.22 (Aug 1989): 343–352.https://doi.org/10.1016/0014-4827(89)90395-9David Epel An Ode to Edward Chambers: Linkages of Transport, Calcium and pH to Sea Urchin Egg Arousal at Fertilization, (Jan 1989): 271–284.https://doi.org/10.1007/978-1-4757-0881-3_14B. Ciapa, G. de Renzis, J. P. Girard, P. Payan Sodium-potassium exchange in sea urchin egg. I. Kinetic and biochemical characterization at fertilization, Journal of Cellular Physiology 121, no.11 (Oct 1984): 235–242.https://doi.org/10.1002/jcp.1041210129Bennett M. Shapiro, E.M. Eddy When Sperm Meets Egg: Biochemical Mechanisms of Gamete Interaction, (Jan 1980): 257–302.https://doi.org/10.1016/S0074-7696(08)61976-2Shun-ichi Miyazaki Fast polyspermy block and activation potential, Developmental Biology 70, no.22 (Jun 1979): 341–354.https://doi.org/10.1016/0012-1606(79)90032-0Laurinda A. Jaffe, Kenneth R. Robinson Membrane potential of the unfertilized sea urchin egg, Developmental Biology 62, no.11 (Jan 1978): 215–228.https://doi.org/10.1016/0012-1606(78)90103-3David Nishioka, Nicholas Cross THE ROLE OF EXTERNAL SODIUM IN SEA URCHIN FERTILIZATION11This work was supported by a grant from the National Science Foundation to Dr. D. Epel., (Jan 1978): 403–413.https://doi.org/10.1016/B978-0-12-217850-4.50039-3Kenneth R. Robinson Potassium is not compartmentalized within the unfertilized sea urchin egg, Developmental Biology 48, no.22 (Feb 1976): 466–472.https://doi.org/10.1016/0012-1606(76)90109-3Edward L. Chambers, Berton C. Pressman, Birgit Rose The activation of sea urchin eggs by the divalent ionophores A23187 and X-537A, Biochemical and Biophysical Research Communications 60, no.11 (Sep 1974): 126–132.https://doi.org/10.1016/0006-291X(74)90181-8R.Douglas Powers, Joseph T. Tupper Some electrophysiological and permeability properties of the mouse egg, Developmental Biology 38, no.22 (Jun 1974): 320–331.https://doi.org/10.1016/0012-1606(74)90010-4Joseph T. Tupper Inhibition of increased potassium permeability following fertilization of the echinoderm embryo: Its relationship to the initiation of protein synthesis and potassium exchangeability, Developmental Biology 38, no.22 (Jun 1974): 332–345.https://doi.org/10.1016/0012-1606(74)90011-6Joseph T. Tupper, R. Douglas Powers Changes in ion permeability and membrane potential during early echinoderm development: Electrophysiological and tracer-flux determinations, Journal of Experimental Zoology 184, no.33 (Jun 1973): 353–363.https://doi.org/10.1002/jez.1401840309Joseph T. Tupper Potassium exchangeability, potassium permeability, and membrane potential: Some observations in relation to protein synthesis in the early echinoderm embryo, Developmental Biology 32, no.11 (May 1973): 140–154.https://doi.org/10.1016/0012-1606(73)90226-1 Bibliography, (Jan 1973): 390–460.https://doi.org/10.1016/B978-0-12-285750-8.50018-0Robert David Allen, L. Jacobsen, J. Joaquin, L.F. Jaffe Ionic concentrations in developingPelvetia eggs, Developmental Biology 27, no.44 (Apr 1972): 538–545.https://doi.org/10.1016/0012-1606(72)90191-1R.M. IVERSON, GERALDINE H. COHEN Polysomes of Sea Urchins: Retention of Integrity, (Jan 1969): 299–313.https://doi.org/10.1016/B978-1-4832-2797-9.50020-0Hector Timourian, Paul C. Denny Activation of protein synthesis in sea-urchin eggs upon fertilization in relation to magnesium and potassium ions, Journal of Experimental Zoology 155, no.11 (Feb 1964): 57–70.https://doi.org/10.1002/jez.1401550105Albert Tyler, Alberto Monroy Changes in rate of transfer of potassium across the membrane upon fertilization of eggs of Arbacia punctulata, Journal of Experimental Zoology 142, no.11 (Oct 1959): 675–690.https://doi.org/10.1002/jez.1401420132F.H.N. Rudenberg The role of the jelly coat in the uptake of calcium by eggs of Arbacia punctulata before and after fertilization, Experimental Cell Research 4, no.11 (Jan 1953): 116–126.https://doi.org/10.1016/0014-4827(53)90194-3

The principal theme of these investigations concerns the inhibited state of mature unfertilized sea urchin eggs with respect to uridine uptake and protein synthesis. Part I demonstrates that unfertilized-eggs are relatively impermeable to uridine. Fertilized eggs, however, develop during the first hour an energy-dependent, uptake mechanism for uridine accumulation. Labeled uridine assimilated by fertilized eggs is recovered as phosphorylated nucleosides, primarily triphosphates. Experiments support the idea that uridine penetration into sea urchin eggs depends upon the phosphorylation of the 5' carbon atom at the cell surface. Tests with puromycin show that protein synthesis is unnecessary for the generation of uridine uptake after fertilization. The evidence favors the view that uridine kinase is sequestered within the unfertilized egg and thus incapable of activity at the cell surface until after fertilization. Parts II and III use biochemical and autoradiographic methods to show that growing oocytes of sea urchins, in contrast to many other organisms, undergo considerable RNA and protein synthesis. Protein synthesis in isolated oocytes occurs throughout the germinal vesicle and cytoplasm and takes place on polyribosomes. RNA synthesis is localized in the nucleolus. Mature eggs, however, synthesize only little protein even in mixed suspensions with oocytes. Long-term maintenance of spawned female sea urchins, after but one injection of labeled uridine, produces ripe unfertilized eggs possessing highly radioactive RNA. The distribution of label in the extracted RNAs is 70-80% ribosomal, 10-20% heterogeneous, and 5-10% soluble. Part IV is an electron microscopic and biochemical examination of RNA-labeled mature unfertilized and fertilized eggs. The findings are correlated with the difference in protein synthesizing activity before and after fertilization. The results show that unfertilized eggs synthesize protein upon RNase-sensitive polyribosomes. The large increase in protein synthesis after fertilization occurs in association with the assembly of additional polyribosomes. Homogenates of unfertilized eggs also possess synthetically inactive, RNase-resistant, ribosomal aggregates. Evidence suggests that trypsin followed by RNase disperses the aggregates. Homogenates of fertilized eggs, however, contain very few RNase-resistant ribosomal aggregates. By forty minutes after fertilization, about 70% of the new protein synthesis can be attributed to the new polyribosomes. The weight of the evidence indicates that the remaining 30% of the stimulation of protein synthesis is due to the activation of masked polyribosomes. Appendix 1 shows that, for unfertilized and fertilized eggs, competition for uptake of amino acids occurs primarily among those belonging to the same charge group. Appendix 2 demonstrates that one amino acid can displace another of the same category from intact eggs both before and after fertilization. By combination of these facts, then, it is possible to achieve greater labeling of egg-proteins than has been previously realized.

In Cartesian diver experiments on the oxygen consumption of oocytes, unfertilized eggs and fertilized eggs from the sea-urchin Psammechinus miliaris and the starfish Asterias glacialis it was found:1. The respiration of ripe Ps. eggs declines rapidly after they have been removed from the ovary into sea water. Starting at a rate that may exceed that of newly fertilized eggs it has thus, after some hours, attained a comparatively low and fairly constant level. The declining curve on kinetical analysis proves to be composed of a monomolecular and a constant part. The respiration curve of Ps. oocytes is of a similar type. In Ast. oocytes and eggs the respiratory decrease, though present, is not so prominent as in Ps. cells (3.112.1, 3.113, 3.122, Fig. 2).2. Though there is a real difference in size between the eggs of the two Ps. phenotypes (the littoral Z-form and the S-form of the depths) no difference is found in the rate of respiration (3.112.2, 3.114).3. Measurements on Ps. oocytes and eggs some hours after removal from the ovary show that the oocytes have only a slightly higher respiration than the eggs. The earlier investigations (Lindahl and Holter, 1941) on Paracentrotus lividus eggs showed that these oocytes maintain a rate of respiration even higher than that of the newly fertilized egg. The findings in Par. might be ascribed to a slow respiration decrease in the oocytes, whereas the decrease is more rapid in the eggs. In Ps. the decrease is about equal in oocytes and eggs (3.112.2, 3.113, 4, Fig. 6).4. In Ast. the primary oocytes respire at a much lower rate than do the secondary ones or the eggs (3.122, 4, Fig. 6).5. In Ps. there is a gradual slight decrease in egg respiration with advancing cytoplasmic maturity (3.113).6. In both Ps. and Ast. the respiration of oocytes in ovarial fluid seems to be of the same order of magnitude as that of oocytes in sea water (3.113, 3.122).7. The shape of the respiration curve in Ps. after fertilization is in full concordance with earlier results obtained with different techniques by Gray (1926) and Lindahl (1939) (3.21, Fig. 3).8. The value of the rise in respiration, that occurs in sea-urchin eggs on fertilization, may entirely depend on where on the slope of the decreasing egg respiration curve fertilization occurs. (This rise is characteristic for sea-urchin eggs and has repeatedly been found by earlier investigators.) It is thought that on natural spawning the rise is rather feebly marked because of early fertilization, and that correspondingly the low level respiration of the unfertilized egg may not be reached (3.21, 4, Figs. 3 and 7).9. In Ast. there is no immediate rise in respiration after fertilization, but there is a gradual rise which exactly resembles the exponential increase in newly fertilized sea-urchin eggs (after the first sudden increase has passed). The rise from the oocyte respiration level to that of the egg will, under natural conditions, not occur outside the ovary, as the cells are shed with broken down nuclear membranes (3.22, 4, Figs. 4, 7 and 8).Cleavage rates are given up to the sixth mitosis for Ps., Ast. and Echinocardium cordatum; hatching time is noted (3.3, Fig. 5).It is discussed whether the decrease in respiration of the unfertilized sea-urchin egg after its removal from the ovary has any possible significance for the biochemical aspects of the sea-urchin egg respiration (4).If the respiration rates found in this investigation are compared on a cell volume basis it is found that the Ast. egg will not fit into the generalized scheme of Whitaker (1933) for marine invertebrate eggs; it is discussed why the Ast. egg respiration is so comparatively low (4, Fig. 6).

High-resolution density-gradient analysis of sea urchin polysomes.

A maternal store of histones in unfertilized sea urchin eggs is demonstrated by two independent criteria. Stored histones are identified by their ability to assemble into chromatin of male pronuclei of fertilized sea urchin eggs in the absence of protein synthesis, suggesting a minimum of at least 25 haploid equivalents for each histone present and functional in the unfertilized egg. In addition, electrophoretic analysis of proteins from acid extracts of unfertilized whole eggs and enucleated merogons reveals protein spots comigrating with cleavage stage histone standards, though not with other histone variants found in later sea urchin development or in sperm. Quantification of the amount of protein per histone spot yields an estimate of several hundred haploid DNA equivalents per egg of stored histone. The identity of some of the putative histones was verified by a highly sensitive immunological technique, involving electrophoretic transfer of proteins from the two-dimensional polyacrylamide gels to nitrocellulose filters. Proteins in amounts less than 2 x 10(-4) micrograms can be detected by this method.

The jelly canal marker of polarity for sea urchin oocytes, eggs, and embryos

The aminoguanide, methylglyoxal bis(guanylhydrazone) (MGBG), was shown to stimulate phosphorylation of RR-SRC, a synthetic protein tyrosine kinase (PTK) substrate, and different levels of tyrosyl phosphorylation of endogenous proteins in a sea urchin egg membrane-cortex preparation. Stimulating protein tyrosine kinase activity in the sea urchin egg stimulated intracellular Ca2+ release, because microinjection of 1-5 mM of MGBG into unfertilized eggs triggered a transient rise in intracellular Ca2+ activity ([Ca2+]i) after a brief latent period. Pretreating eggs with PTK-specific inhibitors, genistein or tyrphostin B42, significantly inhibited the MGBG-induced rise in [Ca2+]i. Methylglyoxal bis(guanylhydrazone) stimulation of PTK activities in the unfertilized sea urchin egg appeared to trigger Ca2+ release through phospholipase C (PLC)-dependent inositol 1,4,5-trisphosphate (InsP3) production. The MGBG-induced Ca2+ response could be suppressed in eggs preloaded with the InsP3 receptor antagonist, heparin, and was reduced in eggs pretreated with U73122, a PLC inhibitor. However, the response was unchanged in eggs treated with nicotinamide, an inhibitor of ADP-ribosyl cyclase, or nifedipine, an inhibitor of nicotinic acid adenine dinucleotide phosphate activity. These results suggest that MGBG may be useful as a chemical agonist of PTK in sea urchin eggs and allow direct testing of the PTK requirement for the transient rise in [Ca2+]i in sea urchin eggs during fertilization. Although genistein was observed to significantly delay the onset, the sperm-induced Ca2+ response in PTK inhibitor-loaded eggs otherwise appeared normal. Therefore, it was concluded that sea urchin eggs contain a PTK-dependent pathway that can mediate intracellular Ca2+ release, but PTK activity does not appear to be required for the fertilization response.