Hexokinase activity from eggs of the sea urchin Arbacia punctulata

1. The hexokinase activity of homogenates of eggs and embryos of the sea urchin Arbacia punctulata has been measured. Expressed as micrograms glucose consumed at 20°C., per hour per milligram of protein the following values were obtained: unfertilized eggs, 67; fertilized eggs, 72; 24 hour plutei, 94; 48 hour plutei, 226. The concentration of the enzyme in the eggs is small and may be calculated to be about 0.001 per cent of the dry weight of unfertilized eggs. 2. The hexokinase activity of the egg homogenate was virtually all recovered in the supernatant fraction when the homogenate was centrifuged at 20,000 x g for 30 minutes and was found to have the following properties: The concentrations for half maximal hexokinase activity with various substrates were, approximately: Glucose, 0,00003 M; fructose, 0.00075; mannose, 0.00007; 2-desoxyglucose, 0.00025. The relative rates of phosphorylation of various sugars by the supernate fraction when saturated with substrate were, approximately: Glucose, 1.0; mannose, 1.2; fructose, 1.8; 2-desoxyglucose, 2.0; glucosamine, 0.6. Adenosinediphosphate and glucose-6-phosphate inhibited the enzyme. No evidence for more than one hexokinase in the Arbacia extracts was found.     

Insemination of immature sea urchin (Arbacia punctulata) eggs

Nuclei from osmotically opened erythrocytes and erythroblasts were injected into nucleated or enucleated Xenopus laevis eggs. Although the cleavage pattern of the recipient eggs which started to divide was normal in about half of the cases, nuclei from erythrocytes injected into nucleated or enucleated eggs never promoted development beyond the early gastrula stage. In contrast, nuclei from osmotically opened erythroblasts injected into enucleated eggs promoted development to early tadpole stages (stages 29–36). Frequently, injection of osmotically broken erythroblasts injected into nonenucleated eggs gave rise to triploid larvae which all died at roughly the same early tadpole stages (29–36). Surprisingly, development did not proceed to the stage of advanced organogenesis (stages 44–47), which is easily reached by gynogenetic haploids: The presence of the haploid genome derived from the egg pronucleus did not significantly improve the developmental capacity. Embryos obtained by single injection of erythrocyte nuclei into nucleated eggs were unable to pass the gastrula stage. To invalidate the interpretation that the observed arrest in development was related to nuclear damage during injection of the recipient eggs, single unbroken erythrocytes and unbroken erythroblasts were transferred into nucleated and enucleated eggs. No cleavage was observed in both classes of eggs injected with unbroken erythrocytes. In contrast, erythroblasts were found to induce cleavage in the recipient eggs at a frequency of about 11%. To ascertain that the nucleus of unbroken erythroblasts participated in development, the 1-nucleolar marker was used. Diploid embryos with only one nucleolus present were found following injection of unbroken erythroblasts into enucleated eggs from 2nu females. Triploid 2nu embryos were detected following injection of (diploid) 1nu erythroblasts into nonenucleated eggs from 2nu females. The most advanced development stages reached by these embryos did not, however, differ from the best results found in the first class of experiments: Nuclei from erythroblasts injected undamaged into nucleated or enucleated eggs never developed into a normal tadpole. Serial transfer experiments were performed using normally gastrulating embryos which had developed, following the injection of 1nu unbroken erythroblasts into recipient eggs. These donors for serial transfer experiments were checked for the presence of the 1nu marker. In addition they had passed through a normally cleaving eight-cell stage. No improvement in developmental capacity as compared to first transfer experiments could be found.     

Cholinephosphotransferase activity during development of the sea urchin, Arbacia punctulata

Cholinephosphotransferase activity was examined during early development of Arbacia punctulata embryos. CMP was rapidly incorporated by enzyme preparation from Arbacia embryos into a compound identified as CDP-choline. Activity was dependent upon the presence of Mg²⁺, Mn²⁺, or Co²⁺, and was maximal in phosphate buffer, pH 7.0, containing 3 × 10^?2M MgCl₂ and 2 × 10^?5M CMP. Addition of ATP or egg lecithin had no effect on enzyme activity. The activity was localized in the 1000 g and 10,000 g pellets, with little or no activity in the microsomal fraction.Upon fertilization, cholinephosphotransferase activity decreased rapidly, detectable changes in activity being observed within 2 min after fertilization. After reaching a minimum activity 2 hr after fertilization, the enzyme activity increased up to the gastrula stage, then decreased once more. Possible interference by competing enzymes was examined and found to be negligible.     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

Calcium-binding modulator protein from the unfertilized egg of the sea urchin Arbacia punctulata

We have purified and partly characterized a calcium-binding protein from the unfertilized egg of the sea urchin Arbacia punctulata. This protein closely resembles the calcium-binding modulator protein of bovine brain in its molecular weight, electrophoretic mobility, amino acid analysis, and peptide map. It activates bovine brain phosphodiesterase in the presence of calcium but has no effect on the phosphodiesterase of the Arbacia egg. Densitometric scanning of acrylamide gels of arbacia egg homogenates shows the modulator protein to represent 0.1% of the total protein of the egg. At 10(-4) M free calcium, the protein binds four calcium ions per 17,000-dalton molecule. We have used a column of rabbit skeletal muscle troponin-I covalently coupled to Sepharose 4B as an affinity column to selectively purify the Arbacia egg calcium-binding protein. This column has also been used to purify bovine brain modulator protein and may prove of general use in isolating similar proteins from other sources. The technique may be particularly helpful when only small quantities of starting material are available.     

Benzohydroxamic acid induces polyspermic fertilization in the sea urchin Arbacia punctulata

Benzohydroxamic acid (BHA) is a competitive inhibitor of the sea urchin sperm peroxidase. We now report that addition of BHA to fertilization cultures of Arbacia punctulata promotes polyspermy. This effect is dose and sperm density dependent. The cortical reaction (elevation of the fertilization envelope) is not retarded by BHA. BHA must be added to the cultures before the eggs complete the cortical reaction at 60 sec post insemination in order to induce polyspermy. Since sea urchin eggs release H2O2 during the cortical reaction at fertilization, these findings support our hypothesis that the sperm peroxidase has a functional role in helping to prevent polyspermy.     

Identification of a sperm receptor on the surface of the eggs of the sea urchin Arbacia punctulata

The possibility that the surface of the egg of the sea urchin Arbacia punctulata contains a species-specific receptor for sperm has been investigated. The extent of fertilization of eggs of A. punctulata, which is proportional to the number of sperm, is unaffected by the presence of either eggs or membranes prepared from eggs of Strongylocentrotus purpuratus. In marked contrast, membranes prepared from eggs of A. punctulata quantitatively inhibit fertilization of A. punctulata eggs by A. punctulata sperm. Several lines of evidence indicate that this inhibition is due to the presence of a membrane-associated glycoprotein that binds to the sperm, thus preventing them from interacting with receptor on the surface of the eggs. First, eggs treated with trypsin are incapable of being fertilized, although they can be activated with the Ca2+ ionophore A23187. Moreover, membranes prepared from eggs pretreated with trypsin do not inhibit fertilization of eggs. Second, receptor isolated in soluble form from surface membranes binds to sperm and thus prevents them from fertilizing eggs; the inhibition by soluble receptor is species-specific. Third, the soluble receptor binds to concanavalin A-Sepharose. Fourth, eggs are incapable of being fertilized if they are pretreated with concanavalin A. The specificity of inhibition, and the affect of trypsin and concanavalin A on intact eggs, suggest that the receptor is a species-specific macromolecule located on the surface of the eggs. The sensitivity of the receptor to trypsin, and its ability to bind to concanavalin A, indicate that it is a glycoprotein.     

Diamide reversibly induces a stress response in the sea urchin, Arbacia punctulata

Sea urchin blastulae were treated with two concentrations (0.54 and 0.72 mM) of diamide, a sulfhydryl oxidant, after hatching. These treatments increased the relative synthesis of one set of embryonic proteins while decreasing that of another. This was demonstrated by quantitating the incorporation of [35S]methionine into polypeptides separated by 2-dimensional polyacrylamide gel electrophoresis (2D PAGE). These shifts were dose dependent and apparently reversible after the embryos had regenerated reduced sulfhydryls. Those proteins showing increased incorporation migrated at the same position by 2D PAGE as heat shock proteins, suggesting that diamide was inducing a stress response. Diamide also caused some developmental aberrations at low frequency, and reversibly inhibited ciliary beating.     

Two-dimensional electrophoretic analysis of major phosphoproteins of the sea urchin, Arbacia punctulata

Phosphoproteins of hatched blastulae, gastrulae, and pluteus larvae of the sea urchin, Arbacia punctulata, were labeled in vivo with [32P]O4 and analysed by 2-dimensional polyacrylamide gel electrophoresis and autoradiography. At least 60 phosphoproteins were resolved. Some of these showed different relative intensities of labeling at the embryonic periods monitored. Some embryonic phosphoproteins were characterized by cell fractionation and by comparing autoradiograms with Coomassie-blue staining patterns and [35S]methionine labeling patterns. Neither actin nor tubulin phosphorylation was detected. No differences in phosphorylation were detected in dissociated and partially reassociated blastula cells relative to each other and to intact embryonic controls.     

Mechanism of univalent antisperm antibody inhibition of fertilization in the sea urchin, Arbacia punctulata

Univalent antisperm antibodies (IFab) markedly inhibited the fertilizing capacity of sperm when tested on intact, dejellied, and "demembranated" Arbacia punctulata eggs. Sperm motility and egg jelly penetration were not affected by IFab. Antifertilizin was excluded as the essential sperm antigen involved in the fertilization-inhibiting action. Sperm pretreated with IFab did not bind to the surfaces of either dejellied or demembranated eggs, whereas control globulin (CFab) and seawater-pretreated sperm bound to such eggs in high numbers. Electron microscopy showed that IFab-treated sperm failed to undergo the acrosome reaction. This excluded "bindin" as the essential antigen. Inhibition of fertilization by IFab was reversed or bypassed by artificial induction of the acrosome reaction with ionophore A23187. It is concluded that univalent antisperm antibody treatment inhibits the fertilizing capacity of sperm by preventing a sperm-egg interaction that results in the acrosome reaction; consequently, attachment of the sperm to the egg is prevented.     

Temperature induced isozyme variants in individuals of the sea urchin, Arbacia punctulata.

1. The technique of microacrylamide slab gel electrophoresis was used to repeatedly monitor the qualitative isozyme composition in the tube feet of the sea urchin, Arbacia punctulata exposed to different temperature regimes. 2. Four enzyme systems were assayed. Three (MDH, HK, and ACPH) were monomorphic for each animal studied and no change in band migration pattern was observed. Banding patterns for the fourth system (EST) were observed to vary in the same individual. 3. The reversible induction of esterase variants in the tube feet of the same individual is reported. 4. The change was temperature dependent, and a period of 7-14 days acclimation was required to induce the pattern alteration.     

A hydrogen peroxide block to polyspermy in the sea urchin Arbacia punctulata

Fertilization by more than one sperm in sea urchins inevitably leads to uneven division and death of the embryo. We provide evidence for a block against this polyspermy involving the hydrogen peroxide release by the egg during fertilization that is triggered by entry of the successful sperm. Polyspermy in 100% of fertilized eggs was demonstrated when catalase was added to destroy hydrogen peroxide immediately after fertilization. Soybean trypsin inhibitor, another polyspermic agent, is shown to prevent the formation of hydrogen peroxide in the fertilized egg. This suggests that the protease released from egg cortical granules during fertilization plays a role in the hydrogen peroxide generating system.     

Organization of microfilaments in sea urchin (Arbacia punctulata) eggs at fertilization: Effects of cytochalasin B

Investigations were carried out to examine more closely the aggregations of microfilaments associated with the elongation of microvilli and formation of fertilization cones and the effects of cytochalasin B (CB) on these processes in Arbacia eggs following insemination. At 1 to 5 min postinsemination fertilized eggs were treated with 1–10 μg/ml CB and then prepared for electron microscopy at periodic intervals. Examination of CB-treated and untreated specimens demonstrated that: (1) Reorganization of the egg's microvilli took place soon after insemination; this process, as well as formation of fertilization cones, was correlated with the appearance of fascicles of microfilaments. (2) CB inhibited the formation of fertilization cones and the elongation of microvilli. Bundles of microfilaments were not observed in CB-treated zygotes. (3) CB prevented the normal movements (rotation) of the incorporating spermatozoon into the egg cortex but did not inhibit the migration or fusion of the male and female pronuclei.