Purpuratus Eggs Research Articles

Scanning electron microscope studies of sea urchin fertilization I. Eggs with vitelline layers

The surface coats of sea urchin eggs and the events of fertilization which take place on these surfaces were examined with the scanning electron microscope (SEM). Gametes of Stronglyocentrotus purpuratus and Lytechinus pictus were considered in detail; eggs of seven other echinoids were examined for comparative purposes. Jelly coats, preserved by varying the pH of fixation, were found to vary in morphology and solubility properties between species. The vitelline layers of the nine echinoids are characterized by arrays of projections which are impressions of cytoplasmic microvilli in the vitelline layer. After sperm bind to the egg surface via the acrosomal process, fine filaments, apparently an egg response to insemination, further connect some sperm heads and tails to the egg. The cortical reactions spread out as a wave from where the fertilizing sperm fused with the egg; separation of the vitelline layer proceeds as a smooth wave from S. purpuratus eggs and as a series of localized separations in L. pictus eggs. The fertilization membranes of S. purpuratus and Allocentrotus fragilis zygotes are expanded replicas of their respective vitelline layers, suggesting that fertilization membranes are formed by an unfolding of the vitelline layer.

Properties of tubulin in unfertilized sea urchin eggs. Quantitation and characterization by the colchicine-binding reaction.

The colchicine-binding assay was used to quantitate the tubulin concentration in unfertilized Strongylocentrotus purpuratus eggs and to characterize pharmacological properties of this tubulin. Specificity of colchicine binding to tubulin was demonstrated by apparent first-order decay colchicine-binding activity with stabilization by vinblastine sulfate, time and temperature dependence of the reaction, competitive inhibition by podophyllotoxin, and lack of effect of lumicolchicine. The results demonstrate that the minimum tubulin concentration in the unfertilized egg is 2.71 mg per milliliter or 5.0% of the total soluble cell protein. Binding constants and decay rates were determined at six different temperatures between 8 degrees C and 37 degrees C, and the thermodynamic parameters of the reaction were calculated. delta H0=6.6 kcal/mol, delta S0=46.5 eu, and, at 13 degrees C, delta G=-6.7 kcal/mol. The association constants obtained were similar to those of isolated sea urchin egg vinblastine paracrystals (Bryan, J. 1972. Biochemistry. 11:2611-2616) but approximately 10 times lower than that obtained for purified chick embryo brain tubulin at 37 degrees C (Wilson, L.J.R. Bamburg, S.B. Mizel, L. Grisham, and K. Creswell. 1974. Fed Proc. 33:158-166). Therefore, the lower binding constants for colchicine in tubulin-vinblastine paracrystals are not due to the paracrystalline organization of the tubulin, but are properties of the sea urchin egg tubulin itself.

Heterogeneous distribution of “lysosomal” hydrolases in yolk platelets isolated from unfertilized sea urchin eggs by zonal centrifugation

Three typical “lysosomal” glycosidases, α- L-fucosidase, N-acetyl glucosaminidase and N-acetyl galactosaminidase, were localized within the yolk platelets of unfertilized Strongylocentrotus purpuratus eggs. Homogenates of eggs were fractionated by rate-zonal centrifugation, and the isolated particles were subjected to integrated biochemical and morphological (electron microscopic) analysis. Enzymatic markers were used to determine the distribution of mitochondria (cytochrome oxidase), yolk platelets (acid nitrophenyl phosphatase), and cortical granules (β-1,3 glucanase) in the sucrose density gradient. Yolk platelets were isolated in a high state of purity, with contamination by mitochondria and cortical granules at trace levels. Enzymatic heterogeneity exists within the yolk platelet population. Acid nitrophenyl phosphatase and α- l-fucosidase activities appear to be uniformly distributed within all the yolk platelets, while N-acetyl glucosaminidase and galactosaminidase activities appear to be preferentially distributed within the slower sedimenting sub-population of yolk platelets. Another band of hexosaminidase containing particles sedimented slightly slower than the bulk of the yolk platelets, coincident with the mitochondria. The acid hydrolases packaged in the yolk platelets may participate in the mobilization of yolk material after fertilization. The yolk platelet thus appears to be a highly complex and structured “lysosome-like” storage organelle.

Is calcium ionophore a universal activator for unfertilised eggs?

WE have previously exposed sea urchin eggs to micromolar amounts of the divalent transporting ionophore A23187, and observed every parameter of normal fertilisation. The cortical reaction with elevation of the fertilisation membrane, the plasma membrane conductance changes, the respiratory burst, and increases in protein and DNA synthesis were all initiated in the usual fashion1. These activations of Lytechinus pictus and Strongylocentrotus purpuratus eggs were independent of the ionic composition of the external solutions1. A23187 seemed to act by releasing intracellular Ca2+, for eggs preloaded with 45Ca showed a twentyfold increase in 45Ca-efflux when activated by ionophore or fertilisation1. Measurements of free and bound calcium and magnesium in homogenates of unfertilised eggs showed that most of the Mg2+ was already available in soluble form, whereas the Ca2+ was sequestered but available for release1. After we found that 50 µM A23187 could activate eggs of the mollusc Acmaea insessa and the protochordate Ciona intestinalis to undergo several abnormal cleavages, we wondered whether ionophore activation might be a general phenomenon. To our knowledge artificial parthenogenesis has not been obtained with Acmaea and Ciona eggs by other methods. We have now found that calcium ionophore activation is common to several species widely separated phylogenetically.

A comparison of 5-bromodeoxyuridine and thymidine incorporation into fertilized sea urchin eggs

3H-Thymidine ( 3H-TdR) and 3H-5-bromo-2-deoxyuridine ( 3H-BUdR) are first incorporated into newly fertilized Strongylocentrotus purpuratus eggs 30 min after fertilization, which is about the time of pronuclear fusion. An initial incorporation of 3H-BUdR immediately following fertilization has been previously reported. This was found to be due to a radioactive impurity present in samples of 3H-BUdR stored in aqueous solutions. 3H-TdR stored in water, or 3HBUdR stored in 50% ethanol, did not contain this impurity.

A trypsin-like proteinase localized in cortical granules isolated from unfertilized sea urchin eggs by zonal centrifugation. Role of the enzyme in fertilization

A trypsin-like proteinase was localized within a single subcellular compartment of unfertilized Strongylocentrotus purpuratus eggs, the cortical granules. Homogenates of eggs were fractionated by rate-zonal centrifugation. Enzymatic markers were used to determine the distribution of mitochondria (cytochrome oxidase), yolk platelets (acid nitrophenyl phosphatase), and cortical granules (β-1, 3-glucanase) in the sucrose density gradient. A bimodal distribution pattern was obtained for aryl esterase activity (substrate: β-naphthyl acetate), with one peak in the microsomal and the other in the cortical granule fractions. The cortical granule enzyme was characterized as a trypsin-like proteinase, since it also hydrolyzed another typical tryptic substrate α- N-benzoyl- l-arginine ethyl ester and was completely inactivated by soybean trypsin inhibitor (SBTI). The aryl esterase activity in the microsomal fractions was not inhibited by SBTI, while 50% of the total aryl esterase activity in the original egg homogenate was inactivated by SBTI. The identity of the enzyme(s) responsible for the aryl esterase activity associated with the microsomal particles is unknown at present. The cortical granule proteinase functions in the elevation of the fertilization membrane and establishment of the block to polyspermy at fertilization. Arbacia punctulata eggs inseminated in the presence of trypsin inhibitors, SBTI or tosyl lysine chloromethyl ketone (TLCK), failed to elevate normal fertilization membranes and became heavily polyspermic. On the basis of these results and observations made by other investigators with a wide variety of biological systems, it is proposed that trypsin-like proteinases function in the discharge of secretory granules from all types of cells.

Nucleotide phosphatase in Strongylocentrotus purpuratus eggs. II. Reactivation, substrate specificity, activators, and inhibitors.

AbstractA medullary nucleotide phosphatase activity can be demon‐strated in fixed mature S. purpuratus eggs. However, this enzymatic activity is lost from eggs stored for more than four days in a Michaelis buffer prepared in distilled water. The activity can be reinstituted by overnight incubation in sea water or solutions of its major cations, prior to reacting for enzymatic activity.The substrate characteristics of this nucleotide phosphatase system indicate that the activity is not due to nonspecific acid or alkaline phosphatases, organic or inorganic pyrophosphatases, or hexose phosphatases. The activity is present with ATP and other nucleoside tri‐, di‐, and monophosphates and is most accurately labelled as a nucleotide phosphatase.The effects of various selected enzymatic activators and inhibitors upon the nucleotide phosphatase activity, demonstrated with ATP as substrate, are also presented. This activity is inhibited by L‐cysteine, glutathione, sodium fluoride, sodium tartrate, and zinc chloride; not inhibited by p‐chloromercuribenzoic and mersalyl acids; and stimulated by 2,4‐dinitrophenol, sodium azide, iodo‐acetamide, and ouabain. These data indicate some similarities with other phosphatases, but there are discriminating differences which label the enzyme system as a distinct entity.

Nucleotide phosphatase in Strongylocentrotus purpuratus eggs. I. Localization.

AbstractThe demonstration of nucleotide phosphatase activity in mature S. purpuratus eggs is reproducible with the methods used. A Mg + + sensitive activity is demonstrated with ATP as substrate. This activity is present throughout the medulloplasm and only occasionally in the cortex. The activity is lost from eggs stored for more than five days after fixation. In whole mounts of mature eggs, the activity in the medulloplasm suggests many possible subcellular localizations. Localization of the activity to subcellular structures in the medulloplasm was accomplished by density‐gradient centrifugation of the mature eggs. By this method nucleotide phosphatase activity was present in the fractions containing yolk granules and endoplasmic reticulum and was not associated with the lipid droplets, nuclei, Golgi complexes, atypical tubular endoplasmic reticulum, heavy bodies, annulate lamellae, or mitochondria.

On the macromolecular composition of the jelly coat from Strongylocentrotus purpuratus eggs

The number of macromolecular components present in the egg jelly coat of Strongylocentrotus purpuratus was investigated. The material was prepared free from egg contaminants and examined for homogeneity by cellulose acetate electrophoresis, disc gel electrophoresis, ultracentrifugation and immunological studies. The electrophoretic and ultracentrifugal studies showed the presence of at least two components. The two-dimensional immunodiffusion tests using rabbit antiserum against whole jelly coat, exhibited four and immunoelectrophoresis five different antigens. In addition, it was possible to separate two components from gel filtration experiments with Sepharose 4B. It is concluded therefore that the S. purpuratus egg jelly coat contains minimally from two to five macromolecules.

Phospholipid synthesis in cleaving sea urchin eggs: Model for specific membrane assembly

Fertilization of Strongylocentrotus purpuratus eggs stimulates uptake of choline 4-fold; phosphorylation of intracellular choline is increased >20-fold at the same time. Incorporation, predominantly into phosphatidylcholine, is stimulated >10-fold. The figures for Lytechinus pictus eggs are similar. The rate at which phosphatidylcholine becomes labeled in S. purpuratus and L. pictus embryos is relatively low and, in contrast to DNA synthesis, increases only 3- to 4-fold between first cleavage and the hatching blastula stage. There is no indication of phospholipid turnover during this time. Inhibition of DNA synthesis by hydroxyurea reduces the incorporation of choline into phosphatidylcholine without affecting the phosphorylation of free choline. It is concluded that the assembly of new membranes during cleavage is accompanied by physiologically significant de novo synthesis of phospholipid. This system therefore appears to lend itself well to further studies on the biogenesis of specific membranes.

12S and 9S RNA species: Localization and fate during development of strongylocentrotus purpuratus eggs

Polyacrylamide gel electrophoresis of total RNA obtained from Strongylocentrotus purpuratus eggs and embryos have shown this material to contain a 12S and 9S RNA species, among others. These two species, found during early development of these cells, were not present in late prism or plutei stages. The RNA was extracted by cold phenol deproteinization in the presence of bentonite and precipitated by alcohol. RNAse, obtained from the 140 000 g egg-supernatant was tested against total RNA extracted from unfertilized eggs, blastulae, gastrulae and plutei embryos. The amount of nucleotides liberated was identical in all cases. Egg-RNAse, in the presence of plutei RNA, did not give rise to the 12S and 9S RNA species. The eggs and embryos were also separated by preparative ultracentrifugation into the nuclei, yolk, mitochondrial, microsomal and 140 000 g supernatant fractions. Electron microscopy revealed these fractions to be relatively pure, and the 12S and 9S RNA were found exclusively in, or associated with, the yolk granules. These granules, and the two RNA species, could not be observed in the pluteus stage. These results strongly suggest that these RNAs, present in early development, truly represent specific classes of RNA and not products of higher molecular weight degradation.

A 12-S and 9-S RNA species in Strongylocentrotus purpuratus eggs

A 12-S and 9-S RNA species in Strongylocentrotus purpuratus eggs

Serological studies of two hybrid sea urchins

Embryos of the intergeneric hybrid of Lytechinus variegatus eggs and Tripneustes esculentus sperm and the interordinal hybrid of Strongylocentrotus purpuratus eggs and Dendraster excentricus sperm were described, their developmental rate and morphology were compared, and the influence of the paternal nucleus was observed. The antigens present in the unfertilized eggs, mesenchyme blastulae, gastrulae, and larvae of the two hybrids and the four parental species were investigated with the Ouchterlony double diffusion technique, immunoelectrophoresis, and passive cutaneous anaphylaxis in conjunction with specific absorption of the antisera. No paternal antigens or hybrid-specific antigens were detected in extracts of the embryos of either hybrid. Thus, hybrid pluteus larvae of two different crosses which show paternal characteristics are formed without any indication that the zygote nucleus directs the synthesis of any major class of antigenic protein. No molecules of new antigenic specificity were detected in developmental stages of the four parental species investigated. The implications of these findings were discussed.

A change in permeability and uptake of [ 14C]uridine in response to fertilization in Strongylocentrotus purpuratus eggs

1. 1. The extent to which the unfertilized Strongylocentrotus purpuratus egg is permeable to uridine has been investigated by experiments measuring the partitioning of [ 14C]uridine between the inside and outside of the cell while the eggs remained suspended in the radioactive uridine and after the eggs were washed with ice-cold sea water. It was found that very little uridine entered the unfertilized egg. After fertilization, the label became concentrated in the egg and depleted from the environment. The label was not lost from the eggs by washing with sea water. 2. 2. Uridine uptake after fertilization has been in part characterized by its time course, dependence on exogenous uridine concentration and response to 2,4-dinitrophenol. A lag phase, acceleration phase and constant accumulation phase was found in the rate of uridine uptake which reached a plateau within the first hour of development. Once the maximal rate of accumulation was attained, fertilized eggs were shown to be very efficient in concentrating very small amounts of exogenous [ 14C]uridine. As the concentration of uridine was raised beyond 0.5–1.0 μM in the sea water, there was no further increase in the rate of uptake. 2,4-Dinitrophenol-inhibited uridine uptake. At −3° only a very small amount of uridine penetrated into the eggs, and they did not reach equilibrium with the outside concentration, indicating penetration involves more than diffusion. 3. 3. Most of the radioactivity taken up by the fertilized egg remained soluble in ice-cold 0.2 M perchloric acid. No appreciable free [ 14C]uridine was detected inside the fertilized eggs. The soluble radioactivity was identified on the basis of anion-exchange column chromatography to be in the form of phosphorylated nucleosides, primarily triphosphates. 4. 4. The observations are believed to indicate that fertilized eggs accumulate uridine by phosphorylating the nucleoside at the surface of the cell, a process which may be deficient in unfertilized eggs because of lack of phosphate donors or of the necessary enzymes.

THE RESPIRATION OF UNFERTILIZED SEA URCHIN EGGS IN THE PRESENCE OF ANTISERA AGAINST FERTILIZIN

1. Homologous antisera against purified fertilizin, and against extract of fertilized eggs (of Lytechinus pictus) have been shown to temporarily increase the respiratory rate of the unfertilized eggs of L. variegatus. Parallel experiments employing antisera against fertilizin of Strongylocentrotus purpuratus and unfertilized S. purpuratus eggs yielded essentially negative results. Further experimentation is necessary before this apparent difference between the responses of the eggs of these two species to antisera against fertilizin can be resolved.2. Antisera against fertilizin of S. purpuratus were effective in increasing the respiratory rate of unfertilized L. variegatus eggs, indicating the presence of antibody capable of effecting increased respiration.3. Normal sera and antiserum against sperm were without measurable effect on the respiratory rate of L. variegatus eggs.4. None of the eggs treated with antisera against fertilizin showed morphological evidence of activation.

Free Amino Acids in Sea-Urchin Eggs and Embryos.

SummaryThe free amino acids of sea-urchin (Strongylocentrotus purpuratus) eggs and embryos were extracted with 75% ethanol and identified by means of paper chromatography. Extracts of unfertilized eggs contain relatively large amounts of glycine and small amounts of alanine, glutamic acid, valine, threonine, lysine, glutamine, and leucine (or isoleucine) as well as several peptides and unknown ninhydrin-positive substances. The patterns of amino acid and peptide spots on chromatograms of extracts of cleavage stages, blastulae, gastrulae and prism larvae are dissimilar to that of unfertilized eggs.The author is indebted to Mr. Arthur Karler from the Division of Medical Physics for advice on chromatographic technics.

Further experiments in the heterogeneous hybridization of echinoderms

1) InLoeb's method of heterogeneous hybridization in echinoderms, theAsterias ochracea sperm alone is affected by the hyperalkaline sea water. 2) The fertilization ofS. purpuratus eggs byAsterias ochracea sperm may be brought about in normal sea water, provided the eggs are first sensitized by a 4–5 minute treatment with 3/8 M SrCl2 solution. 3) If immediately after fertilization, the eggs are treated for a short time with hypertonic sea water, the vigor of the blastulae is much enhanced, and in case the treatment has lasted 15–40 minutes, they develop into plutei.