AN ACTIN-LIKE PROTEIN OF THE SEA URCHIN EGGS II. DIRECT ISOLATION PROCEDURE

A direct extraction procedure for isolation of an actin-like protein from fresh sea urchin eggs was tried. The actin-like protein was found in the 10,000g supernatant of the egg homogenate. The crude extract obtained from the egg homogenate was purified by ammonium sulfate fractionation and dialysis against an ATP-cysteine solution. The purified actin-like protein occurred as 2.8S globular protein units, which transformed into heavy components of 7–8S and 12–13S on addition of salts. This change was accompanied by a rise in viscosity.     

CORTEX PROTEIN OF SEA URCHIN EGGS I. ITS PURIFICATION AND OTHER PROTEIN COMPONENTS OF THE CORTEX

Isolated cortical hull of the sea urchin egg consisted of a gel layer having 3–4 μ in thickness which could be dispersed with 0.6 m KCl. After removing a protein fraction soluble in 10 m m Tris-HCl buffer (pH 7.0–7.2) containing 1 m m ATP or EDTA and 1 m m GSH, so called KCl-soluble protein of the cortices was obtained. After purifying the "cortex protein", it was homogeneous so far as checked by ultracentrifugation and electrophoresis on a polyacrylamide gel. The cortex protein had a thiol-disulfide exchange activity to Ca-insoluble protein in the ATP-extract of the cortices catalyzed by a transhydrogenase. Neither ovoactin nor actomyosin-like protein was detected in the ATP-extract or the 0.6 m KCl-extract of the cortices respectively. Hyalin was not detected in our KCl-soluble protein fractions of isolated cortices.     

FORCE EXERTED BY THE CLEAVAGE FURROW OF SEA URCHIN EGGS

A drop of ferrofluid injected into the center of a dividing sea urchin egg is deformed into the shape of an hourglass when the cleavage furrow advances. The force applied to the drop is determined from the deformation of the drop and the interfacial tension between the ferrofluid and the protoplasm. The interfacial tension is determined from the deformation of a spherical drop in the protoplasm when a magnetic field is applied, and the force applied to the drop, which is estimated from the deformation by magnetic field of a similar drop in 2 per cent aqueous solution of Triton X-100 and the interfacial tension between the ferrofluid and this solution.
The force applied to the drop in the dividing egg increases during an early stage of cleavage and decreases during a later stage. The force attained a maximum of 9 × 10⁻³ dyne in an egg of Temnopleurus toreumaticus which pinched the drop into two when it divided. Smaller maximum forces, 3.9 × 10⁻³ dyne in the eggs of Temno-pleurus toreumaticus and 2.0 × 10⁻³ dyne in the eggs of Clypeaster japonicus (mean values), were obtained when the furrowing was arrested by the drop. The magnitude of the maximum tension developed in the contractile element located in the furrow cortex is discussed.     

MICROINJECTION OF COLCHICINE INTO SEA URCHIN EGGS

Inhibition of cleavage by colchicine was examined by microinjecting colchicine solution into one of the blastomeres of a sea urchin egg at the two-cell stage. Cleavage was inhibited if the microinjection was made before a critical point prior to the cleavage, whereas cleavage occurred in spite of the destruction of the mitotic apparatus if the microinjection was made after the critical point. The critical point was 10 min before the mid-stage of the cleavage in Clypeaster japonicus and 8 min before the mid-stage in Temnopleurus toreumaticus at 20 ± 1°C, corresponding to the beginning of anaphase. The threshold for the cleavage inhibition of colchicine was estimated to be 3 × 10⁻⁵ M to 3 × 10⁻⁶ M in final concentration in the cell.     

REFRACTIVE INDEX OF THE PROTOPLASM IN SEA URCHIN EGGS*

The distribution of the refractive index (RI) of the protoplasm in sea urchin eggs was determined from the optical path differences at various regions of the cell measured by interference microscopy assuming that the cell structure is symmetrical about the line passing through the center of the cell and that of the nucleus in unfertilized eggs and about the spindle axis in fertilized eggs during mitosis and cleavage. The RI of the cytoplasm in the unfertilized egg was uniform except for the cortical region, which had the RI higher than that of the underlying endoplasm. The RI of the cortex was generally higher than that of the underlying endoplasm, which did not appreciably change during mitosis and cleavage. The RI of the nucleus was lower than that of the cytoplasm. The RI of the mitotic apparatus was lower than that of the surrounding cytoplasm. The fertilization membrane had a thickness of about 0.6 μm in hydrated state and about 25 nm in dried state (mean values). The RI of the perivitelline space was about 0.00015 higher than that of seawater, equivalent to 0.08 g/100 ml of contents.     

THE PYRUVATE METABOLISM OF SEA URCHIN EGGS DURING THE PROCESS OF CELL DIVISION

The eggs of Arbacia and starfish contained about 70 and 25 micrograms of pyruvate per gm. of dry cells respectively. Arbacia eggs utilized added pyruvate, although the O₂ uptake did not increase. On fertilization the utilization of pyruvate increased sevenfold. This pyruvate seems to be metabolized, as in other cells, with diphosphothiamine as coenzyme. The diphosphothiamine content of fertilized and non-fertilized eggs was about 16 micrograms; that of sperm, 30 micrograms. Penetration of sperm into the egg and fertilization with cell division to the pluteus stage did not bring forth appearance of succino-dehydrogenase. The possible mechanism of fertilization and cell division is discussed.     

THE EFFECT OF PENICILLIN ON EGGS OF THE SEA URCHIN,ARBACIA PUNCTULATA

1. Penicillin in the range of concentration from 250 U/ml. to approximately 2650 U/ml. inhibits the rate of cell division of the fertilized sea urchin egg from 0 to 100 per cent. 2. Penicillin in the same range of concentrations has no effect on the oxygen consumption of the unfertilized or the fertilized eggs. 3. Penicillin is bound by some component of the sea urchin egg in amounts sufficiently large to lower the initial concentration, this binding apparently not being related to the inhibitory action.     

MICROPAPILLAE IN SEA URCHIN EGGS*

Sea urchin eggs fixed in a glutaraldehyde-calcium chloride mixture have “micropapillae” with a dense content. If these structures are real, they are likely to be sites where a fusion with the spermatozoon can take place. It is possible however that they represent some kind of preparation artefact without a structural counterpart in the living state.     

MECHANICAL PROPERTIES OF SEA URCHIN EGGS III. VISCO-ELASTICITY OF THE CELL SURFACE

When a sea urchin egg was compressed between two parallel plates, the force required to keep the distance between the plates constant gradually decreased with time. The contours of the compressed egg were different from the contours expected from the assumption that the surface forces are uniform over the entire surface. The surface forces of the egg without deformation computed from the area of the cell surface in contact with the substratum, the density of the egg and its size were 0.02–0.04 dynes/cm in Hemicentrotus pulcherrimus. Larger values were obtained in eggs during compression. Surface forces, which were computed from measurements of the form of the egg and the applied force when the egg was deformed by a rod and a plate supporting the egg, increased as the deformation increased.
From these results, it was concluded that the cell surface is visco-elastic in sea urchin eggs.     

CHANGE IN THE GLYCOGEN CONTENT OF SEA URCHIN EGGS DURING EARLY DEVELOPMENT

During development of eggs of the sea urchins, Pseudocenrotus depressus and Anthocidaris crassispina , the glycogen level is maintained from the time of fertilization to the swimming blastula stage and then decreases rapidly in the early gastrula stage. During development of eggs of Clypeaster japonicus. Hemicentrotus pulcherrimus and Mespilia globulus the glycogen content decreases slowly from the time of fertilization to the mesenchyme blastula stage, and then more rapidly during gastrulation. The amounts of glycogen mobilized in the embryos from the time of fertilization to the morula stage correspond to 67% of the amount of O₂ consumed in Mespilia eggs, 62% in Clypeaster eggs, 30% in Hemicentrotus eggs and 0–4% in Anthocidaris and Pseudocentrotus eggs. The main energy source in early development seems to differ in different species. When eggs and embryos were incubated with [¹⁴C]glucose for 10min, considarable ¹⁴C-radioactivity accumulated in the glycogen fraction. The rate of [¹⁴C]glucose incorporation into glycogen increased gradually during the first 6 hr after fertilization (up to the morula stage), decreases during the next 4 hr (up to the early blastula stage), and then increased again.     

ENZYMES OF THE TRICARBOXYLIC ACID CYCLE IN SEA URCHIN EGGS AND EMBRYOS

Changes in the activity of some enzymes of the tricarboxylic acid cycle during development of sea urchins were investigated. Unfertilized eggs showed substantial activity of citrate synthase, aconitase, NAD- and NADP-specific isocitrate dehydrogenases, fumarase and malate dehydrogenase. During development, the activity of citrate synthase, aconitase, NADP-specific isocitrate dehydrogenase and malate dehydrogenase increases gradually, whereas the activity of fumarase remains rather constant. There is no close correlation between changes in the enzyme activity and the increase in oxygen consumption during development. Citrate synthase, aconitase, NADP-specific isocitrate dehydrogenase are mainly localized in the mitochondrial fraction, whereas fumarase and malate dehydrogenase are present in both mitochondrial and cytosol fractions. The intracellular localization of these enzymes does not change during development. A possible mechanism for the regulation of some enzymes of the tricarboxylic acid cycle in sea urchin eggs is discussed.     

CHANGE IN THE ACTIVITY OF ARYLESTERASE IN SEA URCHIN EGGS FOLLOWING FERTILIZATION

The activity of arylcsterase in sea urchin eggs ( Anthocidaris craxsispina ), increases at 5 min after fertilization to about 1.5-fold that in unfertilized eggs, and decreases at 15 min to a lower level than that in unfertilized eggs. Then the activity of the enzyme increases again. The enzyme activity in unfertilized eggs is enhanced by either fructose 1, 6-diphosphate (FDP) at concentrations between 4 and 10 μM, or guanosine 3', 5'-cyclic monophosphalc (cGMP) at concentrations between 0.1 and 0.3 μM. The activity is detectable in the crude microsomal fraction and also in the supernatant fraction obtained from sea urchin egg homogenates by centrifugation at 105,000 × g for 2 hr.     

THE EFFECT OF SOLUBLE EGG JELLY ON THE FERTILIZABILITY OF ACID-DEJELLIED SEA URCHIN EGGS*

Acid-dejellied Lytechinus pictus eggs bind few sperm and show decreased fertilizability. Addition of solubilized egg jelly increases sperm binding and fertilizability, presumably by increasing the frequency of the acrosome reaction. However, dejellied Strongylocentrotus purpuratus bind more sperm and show increased fertilizability in the complete absence of soluble egg jelly. Addition of soluble egg jelly greatly decreases fertilizability in S. purpuratus. Such species differences may be the basis for the controversy between Lillie and Tyler on the one hand, who believed that egg jelly increased egg fertilizability; while Loeb and Hagström on the other hand, believed jelly had no effect on, or actually decreased egg fertilizability. ¹²⁵I-labeling of dejellied S. purpuratus egg surfaces and immunofluorescent studies show that egg jelly persists on the surfaces of acid-dejellied eggs. Egg jelly appears to be a non-removable component of the vitelline layer of this species.     

PRODUCTION AND UTILIZATION OF GLUCOSE 1-PHOSPHATE IN THE EGGS OF THE SEA URCHIN ANTHOCIDARIS CRASSISPINA

Concentrations of G1P, G6P, UDPG, UTP and PPi were measured in the eggs of the sea urchin, Anthocidaris crassispina. Activities of phosphorylase a (EC 2.4.1.1), phosphoglucomutase (EC 2.7.5.1), UDPG pyrophosphorylase (EC 2.7.7.9) and pyrophosphatase (EC 3.6.1.1) were also estimated. Levels of G1P and G6P increase following fertilization, but concentrations of UDPG and UTP in unfertilized eggs are very similar to those in fertilized eggs. PPi is undetectable. In unfertilized and fertilized eggs, the G1P level is very low as compared with the G6P level and is far less than that expected from the equilibrium constant in a reaction catalyzed by phosphoglucomutase. Since the phosphoglucomutase activity is higher by about 20 times than the phosphorylase a activity, G1P is probably produced in the reverse reaction, catalyzed by phosphoglucomutase, rather than in the reaction catalyzed by phosphorylase. The G1P thus produced seems to be utilized thoroughly in the reaction catalyzed by UDPG pyrophosphorylase. The reaction seems to be irreversible and tends to go to UDPG production in sea urchin eggs, since the PPi level is negligible due to high pyrophosphatase activity. The utilization of G1P in the reaction catalyzed by UDPG pyrophosphorylase seems to keep the G1P level low.