Unfertilized Sea Urchin Eggs Research Articles

Identification of a calsequestrin-like protein from sea urchin eggs.

Following studies on calcium transport by isolated smooth endoplasmic reticulum from unfertilized sea urchin eggs (Oberdorf, J. A., Head, J. F., and Kaminer, B. (1986) J. Cell Biol. 102, 2205-2210) we have purified and partially characterized a calsequestrin-like protein from this organelle isolated from eggs from Strongylocentrotus droebachiensis and Arbacia punctulata. Muscle calsequestrin from sarcoplasmic reticulum is well characterized as a calcium storage protein. The egg protein resembles calsequestrin in its behavior in purification steps, electrophoretic mobility, blue staining with Stains-all on polyacrylamide gels, and its calcium binding and amino acid composition. Purification was attained with DEAE-cellulose and hydroxyapatite chromatography. The egg protein Mr of 58,000 in the Laemmli gel system is reduced to 54,000 under Weber-Osborn (neutral) conditions, thus showing a pH dependence in its mobility, although less than occurs with muscle calsequestrins. 25% of its amino acids are acidic and 10% basic. It binds 309 nmol of Ca2+/mg of protein, within the range reported for cardiac calsequestrin. Antigenically, the sea urchin egg protein is related to cardiac calsequestrin capable of binding anti-cardiac calsequestrin antibody.

Translational regulation in sea urchin eggs: A complex interaction of biochemical and physiological regulatory mechanisms

AbstractThe unfertilized sea urchin egg is a metabolically quiescent cell. Fertilization results in the activation of a variety of metabolic and biosynthetic pathways, including a 20‐ to 40‐fold increase in the rate of protein synthesis by 2 h after fertilization. This increase is regulated at a purely translational level without the need for new transcription. The greatest part of this increase is due to the translation of stored maternal mRNAs which were not translated in the egg. There is also a 2‐to 3‐fold increase in the peptide elongation rate. The molecular and physiological mechanisms responsible for this activation process are beginning to be understood, and turn out to be much more complex than was anticipated.

Dynein isoforms in sea urchin eggs.

Biochemical and immunological analysis of unfertilized sea urchin eggs has revealed the presence of at least two distinct isoforms of cytoplasmic dyneins, one soluble and the other microtubule-associated. The soluble enzyme is a 20 S particle with a MgATPase activity that can be activated 5-fold by nonionic detergents. It contains heavy chain polypeptides that 1) comigrate with the dynein heavy chains of embryonic cilia; 2) cross-react with antibodies against flagellar dynein; and 3) are cleaved by UV irradiation in the presence of MgATP and sodium vanadate into specific peptide fragments. The soluble egg dynein is, therefore, closely related to axonemal dynein and may be a ciliary precursor. Egg microtubule preparations contain a distinct dynein-like polypeptide, previously designated HMr-3 (Scholey, J.M., Neighbors, B., McIntosh, J.R., and Salmon, E.D. (1984) J. Biol Chem. 259, 6516-6525). HMr-3 binds microtubules in an ATP-sensitive fashion; it sediments at 20 S on sucrose density gradients, and it is susceptible to vanadate-sensitized UV cleavage. However, HMr-3 can be distinguished from the soluble cytoplasmic dynein on the basis of its weak cross-reactivity with antiflagellar dynein antibodies, its heavy chain composition on high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its low specific ATPase activity, and the molecular weight of its vanadate-induced UV cleavage fragments. HMr-3 may represent a dynein-like polypeptide that is distinct from the pool of ciliary dynein precursors.

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.

Ionic strength-dependent isoforms of sea urchin egg dynein.

Unfertilized sea urchin eggs provide a reservoir of molecules which later are involved in microtubule-mediated movements during embryonic development. Among these molecules is egg dynein, which has been isolated in two forms, 20 S and 12 S. Evidence obtained previously from our laboratory indicates that 20 S dynein is a latent activity precursor of ciliary dynein. In contrast, others have suggested that 12 S egg dynein functions in the mitotic apparatus. It is therefore important to determine the relationship between these egg dyneins. Here we demonstrate that the sedimentation velocity of the egg dynein is dependent on the ionic strength of the extraction conditions. The 20 S dynein is obtained with low ionic strength extraction, and the 12 S form is obtained in high salt (0.6 M KCl). The 20 S dynein, after collection from a sucrose gradient, can be converted quantitatively to the 12 S form by exposure to salt, and this conversion can be followed over time. Further, the 20 S dynein can be converted entirely to 12 S dynein and then partially reconstituted to a faster sedimenting species. During these conversions, the dynein high Mr heavy chains are always coincident with the MgATPase activity, and antibodies show that the dynein heavy chains of the 20 S, 12 S, and converted species are indistinguishable immunologically. These data suggest that 12 S dynein is an ionic strength-dependent isoform of 20 S dynein that results from a partial dissociation of the 20 S polypeptide complex, similar to the relationship between 12 and 21 S sperm flagellar dynein. If the 20 and 12 S enzymes are isoforms of the same dynein, then there is compelling evidence for only a single dynein in the unfertilized egg, and that dynein is probably a ciliary precursor.

Unfertilized sea urchin eggs contain a discrete cortical shell of actin that is subdivided into two organizational states.

Changes in the distribution and organizational state of actin in the cortex of echinoderm eggs are believed to be important events following fertilization. To examine the initial distribution and form of actin in unfertilized eggs, we have adapted immunogold-labeling procedures for use with eggs of Strongylocentrotus purpuratus. Using these procedures, as well as fluorescence microscopy, we have revealed a discrete 1-micron-thick concentrated shell of actin in the unfertilized egg cortex. This actin is located in the short surface projections of unfertilized eggs and around the cortical granules in a manner that suggests it is associated with the cortical granule surface. The actin in the short surface projections appears to be organized into filaments. However, most if not all of the actin surrounding the cortical granules is organized in a form that does not bind phalloidin, even though it is accessible to actin antibody. The lack of phalloidin binding is consistent with either the presence of nonfilamentous actin associated with the cortical granules or the masking of actin-filament phalloidin-binding sites by some cellular actin-binding component. In addition to the concentrated shell of actin found in the cortex, actin was also found to be concentrated in the nuclei of unfertilized eggs.

Inhibitor of eukaryotic initiation factor 4F activity in unfertilized sea urchin eggs.

Extracts from unfertilized sea urchin eggs contain an inhibitor of translation that inhibits protein synthesis in cell-free translation systems from sea urchin embryos or rabbit reticulocytes. The inhibitory effects of egg extracts can be reversed by the addition of mammalian eukaryotic initiation factor 4F (eIF-4F) in both sea urchin embryo and reticulocyte systems, suggesting that the inhibitor inactivates this initiation factor. The accumulated data suggest that the ability of eIF-4F to recycle may be compromised. The addition of eIF-4F to cell-free translation systems from unfertilized sea urchin eggs also stimulates protein synthesis. However, the stimulation does not increase protein synthetic activity in the egg cell-free translation system to the levels observed in those produced from 2-hr embryos. This suggests that, although the unfertilized egg contains an inhibitor of eIF-4F and reduced levels of eIF-4F activity, inactivation of this component is only one of the factors involved in the low rate of maternal mRNA utilization found prior to fertilization.

Low ionic strength solubility of myosin in sea urchin egg extracts is mediated by a myosin-binding protein.

We identify a novel myosin-binding protein, designated 53K, which appears to mediate the low ionic strength solubility of myosin in extracts of unfertilized sea urchin eggs. The protein possesses a subunit molecular mass on SDS-PAGE of 53 kD, an S value of 7, may be organized into disulfide-linked oligomers, and is associated with myosin in egg extracts. Both myosin and 53K co-precipitate from extract upon the addition of nucleoside triphosphates and co-sediment with an S value of 24 by sedimentation velocity centrifugation. Myosin in extracts not associated with 53K has an S value of 10. Further, myosin can be immunoprecipitated from extract with antibody to 53K and the 53K in extracts binds to a myosin affinity column. When extract is depleted of 53K, a majority of the myosin precipitates out of extract in a nucleotide-independent manner. Whereas purified myosin precipitates in the absence of nucleotide when recombined with dialysis buffer or myosin-depleted extract, reconstituting 53K and myosin before addition to buffer or myosin-depleted extract partially restores the low ionic strength solubility demonstrated by myosin in fresh egg extracts. The 53-kD protein may represent a new class of authentic myosin-binding proteins that may regulate the supramolecular organization of myosin in nonmuscle cells.

Temperature and pH govern the self-assembly of microtubules from unfertilized sea-urchin egg extracts.

A new method for microtubule purification from unfertilized sea-urchin eggs was developed in order to obtain large quantities of calcium- and cold-labile microtubules that contained microtubule-associated components important for mitosis. By taking into consideration the pH, ionic composition of egg cytoplasm, and the physiological temperature for growth of the Pacific coast sea-urchin Strongylocentrotus purpuratus, methods were developed for the assembly of intact microtubules directly from unfertilized egg extracts. The microtubules obtained by cycles of temperature-dependent assembly and disassembly are composed of tubulin and abundant microtubule-associated proteins. These microtubules are cold- and calcium-labile and assemble at a critical protein concentration of 0.11 mg ml-1 at 24 degrees C. The yield of microtubule protein obtained by this new method is equivalent to that obtained with taxol (6-8 mg/20 ml packed eggs). Microtubules that have been fixed and prepared for electron microscopy are decorated with large, globular projections that are attached to the microtubule by thin stalks.

Deformation analyses in cell and developmental biology. Part II--Mechanical experiments on cells.

This study employs the finite element approach developed in Part I to analyze mechanical experiments on cells. It views cells as axisymmetric membrane structures containing a body of incompressible material, and models the mechanical contact between a cell and the loading apparatus by a contact algorithm. Since the method is valid for analyzing axisymmetric shell-like bodies with arbitrary shapes, it treates various mechanical experiments on cells in a unified manner. For demonstration purposes, three commonly used mechanical experiments on cells are considered; the compression experiment; the suction (micropipette aspiration) experiment; and the magnetic particle experiment. Based on an estimate of the mechanical property data for unfertilized sea urchin eggs, this analysis method predicts the responses for all three experiments using the same assumptions and approximations. This parallel treatment gives a broad basis for data correlation with experiments. The method also provides insights into mechanical experiments not offered by other approximate methods. For example, it gives the distributions of tensions and stretches on the cell cortex, and suggests the role of friction in the suction experiment.

Localization of sea urchin egg cytoplasmic dynein in mitotic apparatus studied by using a monoclonal antibody against sea urchin sperm flagellar 21S dynein

AbstractA monoclonal antibody against sea urchin (Hemicentrotus pulcherrimus) sperm flagellar 21S dynein was characterized and sued to identify and localized cytoplasmic dynein of sea urchin eggs by the methods of immunoblotting and indirect immunofluorescence microscopy. D57, the monoclonal antibody used in this study, was directed to the Aβ polypeptide of 21S dynein. D57 stained sperm flagella specifically but did not inhibit Mg‐ATPase activity of 21S dynein, its recombination ability with NaCl‐extracted axonemes, or the movement of demembranated sperm. D57 cross‐reacted with sea urchin egg cytoplasmic dynein. High molecular weight cytoplasmic dynein polypeptide which had the same electrophoretic mobility s flagellar dynein. A chains was the only polypeptide that reacted with D57 in the crude extract from unfertilized sea urchin eggs. Indirect immunofluorescence observations showed that the mitotic apparatus was stained most intensely in the frozen sections and lysed eggs. In the mitotic apparatus isolated at metaphase, the half spindles were stained more strongly than the astral regions. The regions near chromosomes in the half spindle appeared to be stained particularly. Staining of the interzone was also observed in the mitotic spindle isolated at anaphase. Comparison of the staining patterns for cytoplasmic dynein with that for tubulin suggested that cytoplasmic dynein was localized where microtubules were densely organized, but its distribution may not necessarily be identical with that of microtubules.

Nucleocytoplasmic Interactions in Morphogenesis

This chapter summarizes and compares the results obtained by merotomy experiments in Acetabularia and sea urchin eggs and discusses the role played by the germinal vesicle (GV) during Xenopus oogenesis and maturation. The role played by the cell nucleus as a whole in morphogenesis is a very old problem; the most direct approach to its solution is merotomy, that is, cutting an egg or a unicellular organism into two halves: nucleate and anucleate. The anucleate fragments of Acetabularia retain a photosynthetic circadian rhythm for several weeks. However, it is the nucleus that sets the clock, as has been shown by experiments in which nucleate and anucleate halves of algae, which were at opposite phases of the cycle, were combined. Unfertilized sea urchin eggs can be cut into two halves (nucleate and anucleate) by high-speed centrifugation in a density gradient. Both nucleate and anucleate fragments can be fertilized or treated with parthenogenetic agents. Oogenesis is the preparation for embryonic development: there is a progressive accumulation of reserve materials (glycogen, lipids, and proteins), of ribosomes, and of about 20,000 different maternal mRNAs. Maturation transforms the fully grown oocyte (1.1–1.2 mm in diameter for Xenopus ) into an egg that, after fertilization, can give rise to an embryo and finally an adult of the same species. The egg is thus a giant totipotent cell. Xenopus eggs are fertilizable when the first polar body has been expelled; they can also be activated by pricking, electrical shock, or treatment with the divalent ions ionophore A23187. As in sea urchin eggs, the main early biochemical changes in activated or fertilized Xenopus eggs are increases in free Ca 2+ and pHi.

Ontogenic activation of a fusion gene introduced into sea urchin eggs.

Regulatory sequences of a sea urchin cytoskeletal actin gene (CyIIIa) were ligated to the bacterial gene coding for chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol O3-acetyltransferase, EC 2.3.1.28) and the construct was injected into unfertilized sea urchin eggs. CAT activity is detected at the early blastula stage, when transcripts of the endogenous CyIIIa gene normally appear. Our measurements show that during activation the amount of CAT enzyme increases at least 100-fold; that there are present in late blastula stage embryos about 5 X 10(5) molecules of CAT mRNA (i.e., approximately 6 times the number of endogenous CyIIIa mRNAs); and that within the range studied the amount of CAT enzyme produced is independent of the number of CyIIIa-CAT genes incorporated per embryo, probably because the genes are present in excess of factors required for their activation. Activation of the CyIIIa-CAT construct is seriously inhibited, or abolished, by successive deletions of upstream CyIIIa sequences.

An antiserum to the sea urchin 20 S egg dynein reacts with embryonic ciliary dynein but it does not react with the mitotic apparatus

Unfertilized sea urchin eggs contain one or more dynein-like enzymes which may be able to serve as microtubule translocators during embryonic development. There are at least two interesting possibilities for the function of the egg dynein: the enzyme may be involved in cytoplasmic microtubule movement such as mitotic spindle anaphase motion; or the enzyme may be a stored precursor for the dynein that functions in embryonic cilia, which are expressed and highly motile at the blastula stage of development. In order to determine directly the distribution and possible function of one of the previously described egg dyneins, the latent-activity 20 S egg dynein ( Asai and Wilson, 1985), an antiserum was produced which was highly reactive with the important high M r polypeptides of 20 S dynein. This antiserum reacted in “Western” immunoblots and in dot-blotting experiments with egg dynein and with embryonic ciliary dynein, but it did not react with any component of sperm flagella. Indirect double immunofluorescence microscopy demonstrated that the anti-20 S antiserum could brightly stain embryonic cilia but it did not stain the sperm flagella from the same sea urchin species. Under the same conditions that the antiserum stained cilia, anti-20 S did not stain the mitotic apparatus but it did appear to stain the cortical region of the dividing egg. In a time-course experiment, the antigen reactive with the anti-20 S antiserum gradually accumulated in the developing early sea urchin embryo. The most significant increase in the apparent concentration of the 20 S dynein occurred just prior to embryonic ciliation and during a period when the mitotic activity of the embryo was in decline. These results lead to two conclusions. First, ciliary dynein and sperm flagellar dynein, although derived from very similar organelles and from the same species of sea urchin, are immunologically distinct. Second, the 20 S egg dynein may be a stored precursor of embryonic ciliary dynein and does not appear to be a component of the mitotic apparatus.

DNA synthesis in vitro with an endoplasmic-reticulum-DNA-polymerase complex from unfertilized sea urchin eggs.

An endoplasmic-reticulum-DNA-polymerase complex was prepared from unfertilized sea urchin eggs and its DNA-synthesizing activity was examined using single-stranded DNA of bacteriophage fd as a template. The complex catalyzed the ribonucleotide-dependent DNA synthesis which required dNTPs, NTPs, Mg2+ and single-stranded DNA. The DNA synthesis was sensitive to aphidicolin and N-ethylmaleimide but was resistant to 2',3'-dideoxyribosylthymine 5'-triphosphate (ddTTP) and alpha-amanitin, suggesting the involvement of DNA polymerase alpha. In parallel with the DNA synthesis, a small amount of RNA was synthesized in the presence of 100 micrograms/ml alpha-amanitin. The Km value of ribonucleotides for the RNA synthesis coincided with that for the DNA synthesis, suggesting a correlation between the DNA and RNA syntheses. Labelling of the products with [gamma-32P]ATP followed by DNA digestion with pancreatic DNase I revealed the attachment of an oligoribonucleotide (7-11 bases in length) at the 5' ends of the DNA products. These observations suggest that in DNA synthesis, primer RNA synthesis occurs first, followed by DNA chain elongation. During 1-90-min incubation, the amount of the DNA synthesized increased but the length was not significantly increased. Over 80% of the number of synthesized DNA molecules comprised a single population of short DNA fragments (60-200 bases, on average 120 bases in length) and the number of fragments increased, depending on the incubation time. However, DNA fragments of various sizes (about 100-6000 bases) were synthesized with DNA polymerase alpha solubilized from the endoplasmic-reticulum-DNA-polymerase complex. All this evidence suggests that in vitro, the complex preferentially synthesizes a particular size of short DNA fragments. The significance of the fragments is discussed.

Rapid visual detection of sperm-egg fusion using the DNA-specific fluorochrome Hoechst 33342

When unfertilized sea urchin eggs are pretreated with the bisbenzimide DNA-specific fluorochrome Hoechst 33342, then washed and fertilized, a single sperm bound to the egg surface becomes intensely fluorescent. The location of the fluorescent sperm on the egg surface coincides exactly with the epicenter of the cortical reaction and the site at which the insemination cone subsequently appears. These observations, coupled with studies of eggs treated with quercetin to prevent fusion, as well as eggs made polyspermic by halothane exposure, indicate that the sperm acquires fluorescence as a consequence of fusion with the fluorochrome preloaded egg. Using a modification of this technique, we have found that cytoplasmic continuity between the sperm and egg is established at 4–8 sec after the onset of the sperm-induced conductance increase in the egg.

Characterization of water in unfertilized and fertilized sea urchin eggs

The water in unfertilized and fertilized sea urchin eggs was characterized with a proton nuclear magnetic resonance (NMR) titration method assuming fast proton diffusion (FPD) between water compartments. This method involves stepwise dehydration with sequential T1 relaxation time and water content determinations. The results analyzed by the FPD model give evidence of intracellular water compartments with three different correlation times: 6 X 10(-12) sec (bulk water), 1 X 10(-10) sec (structured water) and about 2 X 10(-9) sec (bound water). Fertilization is accompanied by a substantial increase in bulk water (from 111 to 414 g H2O per 100 g dry mass) and by a decrease in the water of hydration (from 128 g to 56 g per 100 g dry mass). This study shows that 54% of the water in the unfertilized sea urchin egg has motional properties different from bulk water and that this percentage decreases dramatically shortly after fertilization. Most of the change in T1 relaxation rate observed at fertilization can be accounted for by uptake of bulk water associated with elevation of the fertilization membrane.

Calcium uptake and release by isolated cortices and microsomes from the unfertilized egg of the sea urchin Strongylocentrotus droebachiensis.

Isolated cortices from unfertilized sea urchin eggs sequester calcium in an ATP-dependent manner when incubated in a medium containing free calcium levels characteristic of the resting cell (approximately 0.1 microM). This ATP-dependent calcium uptake activity was measured in the presence of 5 mM Na azide to prevent mitochondrial accumulation, was increased by oxalate, and was blocked by 150 microM quercetin and 50 microM vanadate (known inhibitors of calcium uptake into the sarcoplasmic reticulum). Cortical regions preloaded with 45Ca in the presence of ATP were shown to dramatically increase their rate of calcium efflux upon the addition of (a) the calcium ionophore A23187 (10 microM), (b) trifluoperazine (200 microM), (c) concentrations of free calcium that activated cortical granule exocytosis, and (d) the calcium mobilizing agent inositol trisphosphate. This pool of calcium is most likely sequestered in the portion of the egg's endoplasmic reticulum that remains associated with the cortical region during its isolation. We have developed a method for obtaining a high yield of purified microsomal vesicles from whole eggs. This preparation also demonstrates ATP-dependent calcium sequestering activity which increases in the presence of oxalate and has similar sensitivities to calcium transport inhibitors; however, the isolated microsomal vesicles did not show any detectable release of calcium when exposed to inositol trisphosphate.

The effects of a 45 000 molecular weight protein from unfertilized sea urchin eggs and its 1:1 actin complex on actin filaments.

A 45 kDa actin-binding protein (SU45) has been isolated previously from egg extracts of the Hawaiian sea urchin Tripneustes gratilla by DEAE Sephacel, Sephadex G-75 and hydroxyapatite chromatography. Using pyrene-labelled rabbit skeletal muscle actin, we have found that when SU45 is added to actin in the presence of calcium and the salt concentration is increased, the initial rate of actin assembly is accelerated. Moreover, the final polymer concentration is reduced indicating that SU45 caps the preferred end of actin filaments shifting the critical concentration (Cc) to that of the nonpreferred end. Determination of the Cc as a function of the concentration of SU45 gave an apparent KD of 1 nM. Dilution of F-actin to below its Cc, into buffers containing SU45 and Ca2+ resulted in a sharp increase in the rate of depolymerization; reducing the Ca2+ concentration attenuated this effect. Incubation of SU45 with rabbit skeletal muscle G-actin yielded a 1:1 complex which held 45Ca2+ tightly with a dissociation half-time of 10.8 days. By kinetic analyses of assembly in the presence of the SU45-actin complex and dilution-induced disassembly of filaments precapped with complex, we have estimated both the association rate constant (4.0 X 10(4)M-1s-1) and the dissociation rate constant (0.05s-1) for the nonpreferred ends of actin filaments. Finally, dilution of F-actin to below its Cc, into complex in either Ca2+ or EGTA resulted in a much slower depolymerization consistent with a rapid capping of the preferred end by the SU45-actin complex.

Characterization of the sea-urchin egg microtubule-activated ATPase.

We have found that cytoplasmic extracts from unfertilized sea-urchin eggs contain a prominent microtubule-activated ATPase activity. This activity is induced by polymeric tubulin, but not by tubulin subunits. The activity cosediments with taxol-stabilized microtubules in an ATP-independent manner. We have separated the ATPase from cytoplasmic dynein and other ATPases on sucrose gradients. The sedimentation, enzymic and microtubule-binding properties of the microtubule-activated species show it to be distinct from cytoplasmic dynein, myosin and kinesin. Since the major function of microtubules in the early sea-urchin embryo is in mitosis, this enzyme represents a new candidate for a role in spindle motility.