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.     

Egg jelly induces the phosphorylation of histone H3 in spermatozoa of the sea urchin Arbacia punctulata

When spermatozoa of Arbacia punctulata are labeled with 32P and treated with soluble egg jelly, radiolabel is incorporated into histone H3. The time course of labeling correlates with the period of chromatin decondensation of sperm pronuclei in eggs. Phosphorylation is on serine and may result from increased turnover of phosphate on H3. The macromolecular fraction of egg jelly (and not the peptide fraction) is the inducer of H3 phosphorylation. The reaction is dependent on external Ca2+ and is induced by monensin and A23187. H3 phosphorylation is not induced by the phosphodiesterase inhibitor IBMX and relatively high (250 microM) concentrations of the protein kinase inhibitor H8 are needed to block the reaction, suggesting that it is cAMP independent. A surprising finding is that merely diluting the cells into Na+ free media is the most effective method to induce the radiolabeling of H3. These results are in contrast to findings on the egg jelly induced phosphorylation of histone H1 in S. purpuratus spermatozoa. These species differences must reflect the great evolutionary divergence between these two sea urchin species in the mechanism of regulation of the phosphorylation of nuclear proteins during fertilization.     

Chemotaxis of Arbacia punctulata spermatozoa to resact, a peptide from the egg jelly layer

Resact, a peptide of known sequence isolated from the jelly layer of Arbacia punctulata eggs, is a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for millimolar external calcium. A. punctulata spermatozoa do not respond to speract, a peptide isolated from the jelly layer of Strongylocentrotus purpuratus eggs. This is the first report of animal sperm chemotaxis in response to a defined egg-derived molecule.     

Prolonged incubation in seawater induces a DNA-dependent protein phosphorylation activity in Arbacia punctulata eggs

Various protein kinases are activated in eggs in response to fertilization. We have previously shown that the induction of DNA-dependent protein phosphorylation activity in the sea urchin eggs is triggered by fertilization. The present study demonstrates that the activation of a DNA-dependent serine/threonine kinase in unfertilized eggs of Arbacia punctulata can be achieved without fertilization. Prolonged incubation in seawater resulted in the activation of the eggs with concomitant induction of DNA-dependent protein phosphorylation activity. The activated eggs when fertilized show a slight increase in the phosphorylation activity 10-min post-insemination. The activity gradually declines as the first and second cleavages proceed. The cytoplasmic extracts of the blastulae, gastrulae, and plutei lack the enzyme activity. These findings reveal that not only fertilization but also egg activation serves as a signal for the induction of a DNA-dependent protein phosphorylation activity in sea urchin eggs suggesting that sperm-entry is not required for the induction of the enzyme activity.     

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.     

Glucose-6-phosphate and 6-phosphogluconate dehydrogenases from eggs of the sea urchin,Arbacia punctulata

1. Glucose-6-phosphate and 6-phosphogluconate dehydrogenases have been found in homogenates of Arbacia eggs; 95 per cent of the activity toward each substrate is recovered in the supernatant fraction after centrifuging at 20,000 g for 30 minutes. 2. With glucose-6-phosphate as substrate) the rate of TPN reduction by the supernatant fraction from 1 gm. wet weight unfertilized or fertilized eggs was 1.8 to 3.0 micromoles per minute; this rate is sufficient to support a rate of oxygen consumption 24 times that observed for unfertilized, and 6 times that for fertilized, eggs. Pentose was formed from glucose-6-phosphate at a rate 0.3 to 0.5 that of TPN reduction, when both rates were expressed as micromoles per minute. 3. The concentrations of glucose-6-phosphate and 6-phosphogluconate for half maximal activity were each approximately 0.00004 M for the respective enzymes in the supernatant fraction. Maximal activity toward 6-phosphogluconate was 50 to 60 per cent of that toward glucose-6-phosphate. Glucose-6-phosphate dehydrogenase activity was 50 per cent inhibited in presence of 0.00006 M 2,4,5-trichlorophenol. 4. Reduction of DPN by the supernatant fraction in presence of fructose-1,6-diphosphate and ADP was 0.1 to 0.2 micromoles per minute per gm. wet eggs, indicating that the glycolytic pathway can metabolize glucose-6-phosphate at about 5 per cent the rate at which it can be oxidized by the TPN system from unfertilized or fertilized Arbacia eggs. 5. Phosphoglucomutase, hexose isomerase, and a phosphatase for fructose-1,6-diphosphate also appear to be present in Arbacia eggs.     

Life-history consequences of investment in free-spawned eggs and their accessory coats

The optimal trade-off between offspring size and number can depend on details of the mode of reproduction or development. In marine organisms, broadcast spawning is widespread, and external coats are a common feature of spawned eggs. Egg jelly coats are thought to influence several aspects of fertilization and early development, including the size of the target for sperm, fertilization efficiency, egg suspension time, polyspermy, embryo survival, and fecundity. These costs and benefits of investment in jelly result in trade-offs that can influence optimal reproductive allocation and the evolution of egg size. I develop an optimization model that sequentially incorporates assumptions about the function of egg coats in fertilization. The model predicts large variation in coat size and limited variation in ovum size under a broad range of conditions. Heterogeneity among spawning events further limits the range of ovum sizes predicted to evolve under sperm limitation. In contrast, variation in larval mortality predicts a broad range of optimal ovum sizes that more closely reflects natural variation among broadcast-spawning invertebrates. By decoupling physical and energetic size, egg coats can enhance fertilization, maintain high fecundity, and buffer the evolution of ovum size from variation in spawning conditions.     

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.     

Tetrapyrrol pigments in the test of the echinoid Arbacia lixula

The test of Arbacia lixula contains chlorin e₆ and traces of coproporphyrin I, in addition to the already known naphthaquinone pigments. The possible function of the chlorin is discussed.