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.     

Solubility changes of proteins in sea urchin eggs upon fertilization

The results presented in this paper give evidence of changes in solubility occurring in a protein fraction of the eggs of Arbacia lixula upon fertilization. The electrophoretic analysis indicates that it is only one part of one of the components of the KCl fraction that undergoes the change. However, under some experimental conditions (freezing and thawing of the KCl fraction or extraction of the whole eggs with water at room temperature) a larger portion of the KCl fraction, namely the whole group of components a and b, may be involved and undergoes coagulation. Therefore assuming that the results obtained on the extracts of frozen-dried fertilized eggs do reflect what actually occurs under natural conditions, we must also assume the existence of mechanisms controlling the extent of this change in the living eggs. The fact that in many cases one part or the whole of the sensitive fraction has been found to undergo an increase in solubility may suggest that the process of coagulation discovered by Mirsky is a two-step process. In the first step the sensitive fraction undergoes a change that makes it more soluble and then, when certain conditions are fulfilled, coagulation occurs. An alternative explanation could also be that the coagulated or coagulating fraction is attacked by the proteolytic enzyme that, as shown by Lundblad (1949, 1950), is activated on fertilization. This, however, seems to be less probable, as extraction was always carried out at 0° C. and in as short a time as possible. However, further experiments are needed to decide whether the coagulation of the sensitive fraction is an actual occurrence under natural conditions. The results obtained with the eggs of Arbacia punctulata may cast some doubt on this assumption.     

Cell cycle changes in water properties in sea urchin eggs

This study concerned changes in the motional properties of cellular water during the first cell cycle of fertilized sea urchin eggs (Lytechinus variegatus). There was a significant decrease in proton NMR T1 relaxation time and in cytoplasmic ice crystal growth during mitosis and a significant increase in T1 time and cytoplasmic ice crystal size during cleavage. This was not caused by egg water content changes as reflected by egg volume measurements. Removal of both the fertilization membrane and the hyaline layer shortly after fertilization did not alter the pattern of T1 time changes at mitosis and cleavage as compared to whole eggs; thus, the pattern of T1 time changes was attributed to intracellular events. Treatment of fertilized eggs with cytochalasin B, an inhibitor of actin polymerization, did not block the fall in T1 time at mitosis, but did block cytokinesis and the increase in T1 time, which normally occurred at cleavage. A significant pattern of actin disassembly and reassembly at mitosis and cytokinesis was found by studies on the total amount of monomeric actin (G actin) using the DNase I assay. This led to the hypothesis that the observed changes in T1 time and ice crystal size during the first cell cycle were due to the depolymerization and polymerization of cytoplasmic actin. To test this, the effect of the in vitro polymerization of purified actin on the T1 time and on ice crystal growth was examined. It was concluded that changes in the T1 time and ice crystal growth upon polymerization of actin in vitro resembled the changes seen in vivo. These results suggest that changes in the motional properties of cytoplasmic water during the first cell cycle are due, at least in part, to the state of polymerization of cytoplasmic actin.     

A Ca-activated ATPase in the mitotic apparatus of the sea urchin egg (isolated by a new method)

Steps in a new procedure for isolating the mitotic apparatus from sea urchin eggs (Strongylocentrotus purpuratus) are: (1) Cultivation of the eggs in sea water in which Na is replaced by Li, under which conditions the MA is stabilized in vivo and does not break down at the end of mitosis; (2) storage of the eggs containing the MA in 30% ethanol at −10 °C, preserving isolability and ATPase activity for several months; (3) isolation of the MA in the presence of ethanol and Triton X-100 at +10 °C by selective dispersal of the cytoplasm; (4) purification by washing in 30% ethanol, 0.1% Triton at low temperature. Because of the stabilization of the MA in the Li-sea water, the yield is large.     

Immunofluorescence studies of developmental changes in sea urchin eggs and embryos

By means of indirect immunofluorescence techniques, distinct sea urchin antigens were localized in eggs and embryos (Paracentrotus lividus). The specificity of the method was ascertained from controls in which the specific rabbit anti-sea-urchin sera were substituted by rabbit antiserum to an unrelated antigen (human serum albumin), by normal rabbit serum or by phosphate-buffered saline. The specificity of staining was also evaluated by comparing the different staining patterns obtained either with antisera to whole homogenates of eggs and embryos or with antisera to distinct antigens.     

Peroxidatic activity of catalase in the cortical granules of sea urchin eggs

The presence of peroxidatic activity of catalase in eggs of the sea urchins Hemicentrotus pulcherrimus and Temnopleurus toreumaticus was investigated by the ultrastructural cytochemical techniue and by biochemical assay on homogenates of eggs from before fertilization to the 2-cell stage. Biochemical assays showed that the unfertilized eggs had strong catalase activity whereas fertilized eggs had weak activity owing to the rapid decrease of activity after fertilization. The activity did not change from immediately after fertilization to the 2-cell stage. Cytochemical examination showed that the peroxidatic activity of catalase was mainly localized in the lamellae in the cortical granules. Disintegrated cortical granules with no lamellae and substances in the perivitelline space derived from breakdown of the cortical granules had no peroxidatic activity of catalase.     

Ribosomal proteins of sea urchin eggs

Summary Ribosomal proteins from unfertilized eggs of three sea urchin species, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, and Anthocidaris crassispina, were analyzed. Species-specific differences were observed in the profiles of large subunit proteins on two-dimensional slab gels, though the number of ribosomal proteins and the molecular weights of their counterparts were the same. The small subunit proteins revealed similarities in the electrophoretic profiles and in the phosphorylation patterns among these three species.     

Fertilization kinetics of sea urchin eggs

Eggs and sperm of the sea urchin Paracentrotus lividus of the Mediterranean are used for an in vitro study of fertilization kinetics. The results are analyzed in terms of two models. One of these models assumes that all sperm-egg encounters lead to permanent attachment; the other (less realistically) assumes that sperm continue their random search after an unsuccessful encounter. More than 100 spermatozoa per egg are needed to achieve a fertilization ratio of more than 95%. There are two explanations for this: only 1% of the egg surface is subject to fertilization, or only 1% of spermatozoa are intrinsically able to fertilize. In the same context, chemotactic attraction and the role of the jelly are discussed. Comparison with earlier work of Rothschild and Swann and of Hultin and Hagström clarifies some discrepancies between and within these papers.     

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.     

On the magnesium content in sea urchin eggs and its changes accompanying fertilization

Summary The content of total and bound magnesium of the eggs ofPseudocentrotus depressus andHemicentrotus pulcherrimus was estimated by atomic absorption spectrophotometry. The amount of total magnesium was almost equal (0.128 gmg/g PO₄) in both species and did not change upon fertilization. Bound magnesium decreased in fifteen minutes after fertilization and recovered the original value in further fifteen minutes. The role of magnesium in the fertilization process was discussed with the presence of the experimental results.     

Action of colcemid in sea urchin eggs

The effects of Colcemid, the deacetyl-N-methyl derivative of colchicine, on the eggs of Arbacia punctulata were investigated. Colcemid in concentrations of 2.7 x 10^-5 M or greater blocks syngamy (the fusion of the pronuclei) in these eggs. Although a tenfold decrease in concentration of Colcemid usually permits the pronuclei to fuse, the subsequent division is blocked. In the sea urchin egg, the duration of presyngamy is about 15 min during which time there is no DNA synthesis. However, DNA synthesis is recorded in Colcemid-blocked cells prior to syngamy. Radioautographs of Colcemid-blocked cells which were immersed into thymidine-³H exhibited silver grains above each of the pronuclei. The action of Colcemid on Arbacia eggs is reversible. Nevertheless, exposures to 2.7 x 10^-5 M Colcemid for only 3 min, initiated 5 min after insemination, caused delays of 70 min in subsequent division. In general, cells are more sensitive to Colcemid prior to the time when the mitotic spindle is being assembled than at presyngamy stages. The results are discussed in terms of Colcemid action on pronuclear fusion and cell division.     

Chloride permeability of sea urchin eggs.

We have examined the content and permeability of chloride in sea urchin eggs. After fertilization there is a large increase in the permeability to chloride. We discuss the mechanism underlying this permeability change and the generalized increase in ion permeability observed after fertilization.