INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWING INTERACTION WITH FIXED UNFERTILIZED EGGS

The respiratory rate of spermatozoa of the sea urchins, Anthocidaris crassispina, Clypeaster japonicus and Pseudocentrotus depressus , decreases markedly in the presence of homologous unfertilized eggs fixed with glutaraldehyde. No decrease in the rate of respiration occurs in the presence of fixed fertilized eggs. Fixed unfertilized eggs of different sea urchin species do not cause any change in the rate of sperm respiration. Spermatozoa adhere only to the fixed unfertilized eggs of the same species and are removed by a stirring for 5 min on a magnetic stirrer. The spermatozoa thus removed, are immotile and their respiratory rate is quite lower than that of motile spermatozoa in a control suspension stirred for 5 min. Intact spermatozoa adhere to the fixed eggs, from which the attached spermatozoa have been removed, and the respiratory rate of the spermatozoa also becomes quite low.     

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWIGN INTERACTION WITH FIXED UNFERTILIZED EGGS

The respiratory rate of spermatozoa of the sea urchin, Pseudocentrotus depressus and Hemicentrotus pulcherrimus , became quite low and spermatozoa was immotile, after sperm suspension containing glutaraldehyde-fixed eggs of homologous species was stirred at 20°C for 15 min. The respiratory rate of fresh spermatozoa, introduced to the suspension of immotile spermatozoa thus obtained, was also reduced markedly. The respiration of fresh spermatozoa was not inhibited by adding them to suspension of intact or acrosome reacted spermatozoa. A heat stable and non-dialyzable substance, which inhibited sperm respiration, was removed from the fixed eggs by vigorously stirring the egg suspension for 10 min, when unfertilized eggs were fixed with insufficient amount of glutaraldehyde (10 ml of 1% glutaraldehyde solution to 1 ml egg pellet).     

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.     

SPECIES-SPECIFIC ADHESION OF SPERMATOZOA TO THE SURFACE OF FIXED EGGS IN SEA URCHINS

When the spermatozoa of sea urchins are added to eggs which have been fixed with glutaraldehyde and washed thoroughly, the spermatozoa swarm around the eggs and adhere to the egg surface. The mode of sperm adhesion to the fixed egg is assumed, on the evidence of electron-microscopical studies, to be the same as that of adhesion to the intact egg at the initial stage of normal fertilization. The spermatozoa and fixed eggs of five species of sea urchins were combined and heterologous crosses were studied. Species-specific adhesion of sperm to fixed eggs was clearly demonstrated. There is a direct relationship between the cross-fertilization of living gametes and the binding capacity of spermatozoa and fixed eggs in so far as the employed five species are concerned.     

INITIATION OF CLEAVAGE IN FISH EGGS BY INJECTION OF FLAGELLA OR MICROTUBULES OF SEA URCHIN SPERMATOZOA

Flagella and their microtubules obtained from sea urchin ( Hemicentrotus pulcherrimus ) spermatozoa were injected into unfertilized eggs of the medaka ( Oryzias latipes ) with a micropipette. Upon activation, some of the eggs began the first cleavage with three or more irregular blastomeres, and developed to the morula stage. It is suggested that sperm flagellar microstubule material is one of the cleavage initiation substances.     

CHANGE IN THE ACTIVITY OF PYRUVATE DEHYDROGENASE COMPLEX IN SEA URCHIN EGGS FOLLOWING FERTILIZATION*

The activity of the pyruvate dehydrogenase complex in sea urchin eggs is localized in the crude mitochondrial fraction. The activity of the enzyme complex in the intact mitochondrial fraction of unfertilized eggs is too low to be estimated and is enhanced upon fertilization with a 5-min lag period. The activity of the enzyme complex in unfertilized eggs is enhanced by Ca²⁺at concentrations between 5 × 10^?5 M and 10^?3 M. The activity in fertilized eggs is blocked after incubation with 2 mM ATP, and the block of the activity is also released by Ca²⁺. The blockage of the enzyme complex activity is accompanied by phosphorylation of proteins, and release of the block by Ca²⁺ is concomitantly followed by the dephosphorylation of proteins in the mitochondrial fraction. The enzyme complex in unfertilized eggs will be assumed to be the one inhibited by phosphorylation. The enzyme complex will be activated upon fertilization as a consequence of the dephosphorylation, that is caused by the increase in intracellular concentration of Ca²⁺.     

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.     

LOCALIZATION OF DYNEIN IN SEA URCHIN EGGS DURING CLEAVAGE*

Detection and localization of dynein in cleaving sea urchin eggs were attempted using antidynein serum (prepared against a tryptic fragment of dynein, Fragment A, of sea urchin sperm flagella) and fluorescein conjugated goat antiserum to rabbit γ-globulin. In both unfertilized and newly fertilized eggs, fluorescence was distributed rather uniformly within the cells but was absent from the nuclei. At prophase, intense fluorescence was observed on both sides of nucleus, suggesting accumulation of dynein in developing asters. From metaphase to anaphase, the whole mitotic apparatus (MA) was stained with the exceptions of the chromosomes and pole areas. Fluorescence then again became dispersed within the eggs. Throughout the mitotic process and cytokinesis, the egg cortex including the cleavage furrow was stained intensely, presumably reflecting the presence of dynein in this region. Similar distributions of fluorescence were obtained with the isolated MAs. Neither non-immune serum nor the antiserum to which Fragment A was absorbed stained the eggs. Little staining was obtained with the antiserum against starfish egg myosin. The results, together with the finding that the chromosome motion in the isolated MAs was completely inhibited by anti-dynein serum, but not with the anti-myosin serum, suggest an active role played by a tubulin-dynein system in mitosis.     

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.     

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.     

EFFECT OF TRYPSIN ON THE FERTILIZING CAPACITY OF SEA URCHIN SPERMATOZOA TREATED WITH EGG-WATER*

The effect of trypsin on the fertilizing capacity of spermatozoa was studied with 6 species of sea urchins. Trypsin has no harmful effect on intact spermatozoa. However, spermatozoa which have undergone the acrosome reaction in egg-water lose the fertilizing capacity when treated with trypsin-sea water. Electron- microscopical examination revealed that trypsin does not produce any morphologically noticeable effect on intact spermatozoa, but does dissolve the material covering the acrosomal tubule of the spermatozoa which have undergone the acrosome reaction. It is likely that the loss of this material is closely correlated with the loss of fertilizing capacity of spermatozoa by the trypsin treatment.     

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 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.