Unfertilized Sea Urchin Eggs Research Articles

Stimulation of protein synthesis in unfertilized sea urchin and sand dollar eggs treated with trypsin

Unfertilized sea urchin ( Lytechinus pictus) and sand dollar ( Dendraster excentricus) eggs exposed to trypsin are stimulated to incorporate labelled amino acids into proteins at greatly increased rates. This increase in protein synthesis in trypsin-treated sand dollar eggs is a result of their parthenogenetic activation, whereas the sea urchin eggs do not exhibit any morphological signs of activation from the effects of trypsin. Attempts to artificially activate sea urchin eggs with cyclic adenosine 3′, 5′-monophosphate were unsuccessful.

Reversible response to puromycin and some characteristics of the uptake and use of amino acids by unfertilized sea urchin eggs.

Protein synthesis in embryos and oocytes is irreversibly inhibited when the cells are returned to puromycin-free sea water after previous exposure to puromycin. In unfertilized eggs, on the other hand, it is not irreversibly inhibited. This effect and others are used to show that mature unfertilized eggs are actively engaged in protein synthesis, and that direct assessment of the relative rate of protein synthesis in mature eggs is not hindered by contamination with a small population of very active cells.

Polymerization of actin from sea urchin eggs

Actin was isolated and purified from unfertilized sea urchin eggs ( Pseudocentrotus depressus and Hemicentrotus pulcherrimus), by a method which utilizes its specific binding activity with myosin from rabbit skeletal muscle. The purified actin exists as a monomer (ovo-G-actin) in salt-free solution. The ovo-G-actin has a sedimentation coefficient of 3.0–3.2 S. On the addition of salt (0.1 M KCl), the ovo-G-actin transforms into a heavy component of 25–28 S, which has a higher viscosity value ( η sp/c) of 0.9 – 2.1 dl/g. The heavy component (ovo-F-actin) was revealed by electron microscopy to be a filament having a diameter of 60–80 Å. The ovo-G-actin in salt-free solution also transforms into the ovo-F-actin on the addition of divalent salts, CaCl 2 or MgCl 2.

Methods for removal of the vitelline membrane of sea urchin eggs: I. Use of dithiothreitol (Cleland Reagent)

A simple procedure is described for removing the vitelline membrane of sea urchin eggs. The method is based on the observation that brief incubation of unfertilized sea urchin eggs in 5–10 mM dithiothreitol (DTT or Cleland Reagent) at pH 9.2 effectively removes this membrane. Membrane removal and normal development have been obtained with the eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus. That DTT removes membranes, combined with other observations and experiments, indicates that the structural integrity of the vitelline membrane results in part from disulfide linkages between neighboring protein molecules.

Separation of intact cortical granules from homogenate of unfertilized sea urchin eggs by zonal centrifugation.

Cortical granules are localized in the peripheral cytoplasm of mature, unfertilized eggs. The rupture of these granules constitutes one of the initial morphologically observable responses of the egg to fertilization or parthenogenetic activation. These organelles were separated from homogenates of unfertilized sea urchin eggs ( Strongylocentrotus purpuratus) by fractionation in the A-XII zonal centrifuge using sucrose density gradients constructed in (0.5 M) KCl. Cortical granules were abundant in electron micrographs of the most rapidly sedimenting population of particles. This population was rich in acid nitrophenylphosphatase activity, contained particles which stained metachromatically with toluidine blue and also contained yolk platelets. Acid phosphatase activity was also associated with several populations of slower sedimenting particles. Yolk platelets were widely distributed in the gradient, and were found in all fractions examined. The mitochondria and cytochrome oxidase activity were sedimented a short distance from the starting boundary.

An actin-like protein of the sea urchin eggs: I. Its interaction with myosin from rabbit striated muscle

The existence of an actin-like protein was demonstrated in the extract from acetone-dried powder of unfertilized sea urchin eggs. The ratio of the actin-like protein to the total protein in the extract was 10–13 % as determined by measuring the amounts of the protein which combined with myosin from rabbit muscle. The actin-like protein interacted with rabbit muscle myosin, forming a complex protein which has some characters similar to those of rabbit muscle actomyosin; dissociation and viscosity drop caused by ATP at high salt concentrations, superprecipitation caused by ATP at low salt concentrations, and activation of ATP-ase activity caused by MgCl 2 at low salt concentrations. The actin-like protein has two main bands identical with those of muscle G-actin on polyacrylamide gel electrophoresis.

Reversible inactivation of nicotinamide adenine dinucleotide kinase in extracts of unfertilized sea urchin eggs

NAD kinase activity in extracts of unfertilized sea urchin eggs is lost on dialysis against dilute buffer and can be recovered by incubation with NAD and dithiothreitol.

Regulation of protein synthesis in sea urchin eggs

Treatment of unfertilized sea urchin eggs with cycloheximide results in a 150% increase in polyribosome content, as contrasted with a 10% increase in fertilized eggs under identical conditions. Removal of CO 2 from sea water reversibly stimulates protein synthesis (and polyribosome content) in unfertilized eggs, and changes their response to cycloheximide to correspond with that of fertilized eggs. The stimulation of protein synthesis produced by CO 2 removal is proportional to the initial stimulation of protein synthesis by fertilization in both species studied, and accounts for the major portion of the rate change which occurs in the first 20 minutes after fertilization. Eggs fertilized after stimulation in this way attain the same post-fertilization rate of protein synthesis as control eggs. The evidence presented suggests that the increase in the rate of protein synthesis which accompanies fertilization is due principally to a step up in the rate of chain initiation, and not to the “unmasking” of messenger RNA.

Relative deficiency of intracellular relaxing system observed in presumptive furrowing region in induced cleavage in the centrifuged sea urchin egg

1. 1. Changes in the distribution of the intracellular relaxing system were studied using antibody staining method in unfertilized sea urchin eggs induced to cleave without forming mitotic apparatus by centrifugation and subsequent hypertonic treatment. The relaxing system was found to show a peculiar shift towards centrifugal and centripetal poles, leaving a relaxing system-poor zone between these two poles. Furrowing was always observed to occur just at this relaxing system-poor zone. 2. 2. Microinjection of relaxing granules into ameba cells was also attempted. It was found that the local injction of relaxing granules itself caused no marked change in these ameba cells, but the injection of an extract obtained from relaxing granules had a conspicuous effect on the gel state of ameba cytoplasm. 3. 3. From these results, it may be concluded that the site of experimentally induced furrowing is determined by the distribution pattern of the intracellular relaxing system. Subsequent realization of regional heterogeneity in contractility within cytoplasm might be mediated by release of certain substances, probably of heparin-nature, from the localized relaxing system.

Thymidine kinase, thymidylate kinase and 32P i and [ 14C]thymidine incorporation into DNA during early embryogenesis of the sea urchin

In the early embryogenesis of the sea urchin, the relationship between the activity of thymidine kinase (ATP:thymidine 5′-phosphotransferase, EC 2.7.1.21) and thymidylate kinase (ATP:thymidinemonophosphotransferase, EC 2.7.4.9) and of DNA synthesis was investigated. Thymidine kinase and thymidylate kinase activities were found in the homogenate and in the crude extract of unfertilized sea-urchin eggs and embryos. Maximal activity of the kinases was found in unfertilized eggs and the activity was decreased after fertilization. Thereafter, the activities changed periodically in parallel during the cell cycle and their maxima appeared at a defined stage of the cell cycle. [2- 14C]Thymidine and 32P i incorporation into the DNA fraction was found usually just after each division, with the exception of the first division cycle, which occurred at the stage when pronuclear fusion may take place. DNA synthesis was observed after the appearance of the peak of activity of these two kinases in the division cycles. The DNA synthesis system was not included in one division cycle (the division took place between the time of the appearance of the activity peak of DNA synthesis and that of the two kinases). In the early embryogenesis of the sea urchin, the mechanism of regulation of thymidine kinase activity was investigated and the following results were obtained. Puromycin and ethionine depressed thymidine kinase activity in vivo. Thus, the increase of thymidine kinase activity may be due mainly to enzyme synthesis de novo. Although thymidine kinase was observed to have a low stability, the enzyme had a similar lability in the homogenates of unfertilized eggs and of embryos. Thymidine protected thymidine kinase activity from heat inactivation. dTTP was a feedback inhibitor, but dCDP was not an activator.

Cytoplasmic DNA in the unfertilized sea urchin egg: physical properties of circular mitochondrial DNA and the occurrence of catenated forms.

The mitochondrial DNA in the unfertilized egg of the sea urchin Lytechinus pictus is present in an amount approximately seven times that of the haploid nuclear DNA.(1) The mitochondrial DNA has a higher buoyant density than the nuclear DNA and consists of circular duplex molecules of a uniform size of about 5µ. The circular DNA has been recovered(1) in both the intact (closed) and nicked (open) states characteristic of the circular duplex viral DNA's(2) and the mitochondrial DNA's from birds and mammals.(3, 4)

Protein synthesis by unfertilized eggs of sea urchins.

The known ability of suspensions of unfertilized sea urchin eggs to incorporate amino acids into protein has been examined in four kinds of experiments with Lytechinus pictus. These show: (1) that the incorporation is not attributable to bacteria or other contaminating cells in the suspension but to the eggs themselves; (2) that it is inhibitable by puromycin approximately to the same extent as is protein synthesis in the fertilized eggs; (3) that it occurs upon polysomes; (4) that it is a property of all the eggs and not just a small proportion of unripe ones in the suspension. It is concluded that the ripe unfertilized egg of sea urchins is not synthetically inert but engages in true protein synthesis although at a much lower rate than during oogenesis or after fertilization.

Aminoacyl-sRNA synthetases as possible regulators of protein synthesis in the embryo of the sea urchin Paracentrotus lividus.

The low protein synthetic activity of the unfertilized sea urchin egg is attributed to a translational inhibition.1 Most of the attention has focused on the ribosomes, which have proved to be unable to engage in protein synthesis before fertilization.2' 3 The other components for the translation of messenger ribonucleic acid (mRNA) have not been thoroughly studied; in particular, very little is known as to whether or not transfer ribonucleic acids (tRNA) and aminoacyl-sRNA synthetases (EC 6.1.1) play any role in this process. Suggestions to this effect come from the work of Glisin and Glisin4 and Nemer and Bard.5

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

Abstract Mature unfertilized sea urchin eggs exhibit minimal incorporation of radioactive protein precursors into 5–10% trichloroacetic acid (TCA)-precipitable material (Nakano and Monroy 1958; Hultin 1961; Stafford et al. , 1964; MacKintosh and Bell 1967). One might therefore expect polysomes to be scarce or non-existent in unfertilized eggs. Indeed, recent studies have failed to convincingly demonstrate (by RNase-sensitivity of 260 mμ absorbance and radioactivity) their presence in mature sea urchin eggs before fertilization (Hultin 1964; Malkin et al. 1964; Stafford et al. 1964). The marked rise in protein synthesis which immediately follows fertilization of sea urchin eggs, however, is not mirrored by obvious changes in optical density profiles (Hultin 1964; Malkin et al. 1964; Stafford et al. 1964; Stavy and Gross 1967); nor is polysomal material from older embryos resolved into discrete sedimentation classes (Monroy and Tyler 1963; Hultin 1964; Siekevitz et al. 1966). We now report a method for ribosome-polysome isolation which is consistently effective in yielding sedimentation profiles highly resolved by 260 mμ absorbancy and radioactivity, whether at 15 minutes or 15 hours after fertilization. We also show that the isolation of intact sea urchin polysomes is sensitive to KCl concentrations.

The mechanism of action of colchicine. Colchicine binding to sea urchin eggs and the mitotic apparatus.

Colchicine forms a complex in vivo with a protein present in fertilized or unfertilized sea urchin eggs; similar binding was obtained in vitro with the soluble fraction from egg homogenates. Kinetic parameters and binding equilibrium constant were essentially the same in vivo and in vitro. The binding site protein was shown to have a sedimentation constant of 6S by zone centrifugation. The protein was present in extracts of the isolated mitotic apparatus at a concentration which was several times higher than in whole-egg homogenates. It was extracted from the mitotic apparatus at low ionic strength under conditions which lead to the disappearance of microtubules. No binding could be detected to the 27S protein, previously described by Kane, which is a major protein component of the isolated mitotic apparatus. The properties of the colchicine-bindinG protein, (binding constant, sedimentation constant, Sephadex elution volume) are similar to those obtained with the protein from mammalian cells, sea-urchin sperm tails, and brain tissue, and thus support the conclusion that the protein is a subunit of microtubules.

The stimulation of amino acid incorporation into the protein of unfertilized sea urchin eggs by gamma-irradiation.

AbstractControl and γ‐irradiated sea urchin (Arbacia punctulata) eggs were incubated with H3‐leucine. No incorporation of the amino acid into the proteins of most unirradiated eggs was detectable by autoradiography. All irradiated eggs incorporated the amino acid and the amount of autoradiographic labelling varied greatly from cell to cell.

Stimulation of protein synthesis in unfertilized sea urchin eggs by prior metabolic inhibition

Stimulation of protein synthesis in unfertilized sea urchin eggs by prior metabolic inhibition

Partial activation of intact, unfertilized sea urchin eggs by treatment with polyuridylic acid

Partial activation of intact, unfertilized sea urchin eggs by treatment with polyuridylic acid

A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells.

Covalently closed circular duplex DNA’s are now known to be widespread among living organisms. This DNA structure, originally identified in polyoma viral DNA,1’2 has been assigned to the mitochondrial DNA’s in ox3 and sheep heart,4 in mouse and chicken liver,3 and in unfertilized sea urchin egg.5 The animal viral DNA’s—polyoma, SV40,6 rabbit7 and human8 papilloma—the intracellular forms of the bacterial viral DNA’s—φX174,9, 10 lambda,11, 12 M13,13 and P2214—and a bacterial plasmid DNA, the colicinogenic factor E2,15 have all been shown to exist as closed circular duplexes. Other mitochondrial DNA’s16, 17 and a portion of the DNA from boar sperm18 have been reported to be circular, but as yet have not been shown to be covalently closed.

Radiation-Induced Mitotic Delay in Sea Urchin Eggs Treated with Puromycin and Actinomycin D

The inhibition of protein synthesis by puromycin, which functions as an analog of amino-acyl sRNA (1), appears to result from a release of uncompleted protein chains from those ribosomes engaged in protein synthesis (cf. 2-4). Hultin (5) demonstrated that the compound inhibited amino acid incorporation into proteins in fertilized sea urchin eggs. Furthermore, he found that the cells were blocked specifically in the stage of either the first or second mitotic cycle. This mitotic stage corresponds to the time in early prophase when the centrioles divide and migrate to opposite sides of the nucleus, and the fan of astral rays pushes aside particles, forming a clear streak of cytoplasm adjacent to the nucleus (6). Irradiation prolongs the stage (7, 8), and the first division is delayed only if the cells are irradiated before this stage is clearly visible (9, 10). Since both puromycin and radiation affect the same mitotic stage, possible interactions between the two agents were investigated. Actinomycin D binds to DNA and inhibits the synthesis of RNA (11-13). Gross and Cousineau (14, 15) studied the effects of this drug on the development of sea urchin eggs and found little or no mitotic delay when as much as 96 % of the RNA synthesis was inhibited. However, differentiation beyond the ciliated blastula stage did not occur. Their experiments are consistent with the view that the unfertilized sea urchin egg contains a reservoir of inactive messenger RNA, which participates in protein synthesis after fertilization, and that RNA synthesis per se is not necessary for the early cell divisions (see also 16-19). If irradiation delays division by destroying an essential fraction of the existing RNA or by producing a lesion which requires new messenger RNA for its repair, irradiated cells might prove sensitive to actinomycin D. This possibility was investigated.