The Wave Pattern of Free Calcium Release Upon Fertilization in Medaka and Sand Dollar Eggs

A transient rise in the concentration of Ca²⁺ in the cortex upon fertilization was demonstrated in medaka eggs injected with aequorin. Detection of the aequorin luminescence with an ultra-high sensitivity photonic microscope system revealed a wave of increased Ca²⁺ concentration starting at the site of sperm entry (animal pole) and being propagated along the cortex of the egg toward the antipode. The wave traversed the entire egg surface within 2–3 min. The peak value of the aequorin luminescence, and therefore the peak value of the Ca²⁺ transient, was generally higher at the site of sperm entry than in other regions. The peak values of the luminescence (and therefore of the Ca²⁺ concentration in the cortex) remained fairly constant during propagation of the wave. Microinjection of Ca²⁺ into the cortex also induced a Ca²⁺ wave. When the egg was stimulated by microinjection of Ca²⁺ at the equatorial region, the Ca²⁺ wave was propagated at a fairly constant speed over the egg surface, except at the region near the vegetal pole where the wave was retarded. Simultaneous recording of the Ca²⁺ wave and the wave of cortical change (breakdown of cortical alveoli) in eggs during fertilization revealed that the Ca²⁺ wave preceded the wave of cortical change.
A Ca²⁺ wave was also demonstrated in sand dollar eggs, although due to their smaller size the phenomenon was not as clear as in medaka eggs.     

INDUCTION OF ASTER FORMATION AND CLEAVAGE IN EGGS OF THE SEA URCHIN HEMICENTROTUS PULCHERRIMUS BY INJECTION OF SPERM COMPONENTS*

Studies were made on which components of sperm were able to induce aster formation and cleavage of eggs of the sea urchin Hemicentrotus pulcherrimus. The sperm components were separated by homogenization and centrifugation into the following 3 fractions: the head-midpiece, midpiece and tail. The head-midpiece fraction was then divided into 2 sub-fractions, the centriole sub-fraction and the centriole-free sub-fractions. Each fraction was injected into unfertilized eggs and after 15–30 min the eggs were inseminated. The ability of a fraction or a sub-fraction to induce aster formation and cleavage was deduced from the frequency of multipolar cleavage. The head-midpiece fraction and the centriole sub-fraction were effective in inducing aster formation and cleavage, but the other fractions were not. It was concluded that isolated centrioles from sea urchin sperm act as division centers in the egg.     

Erythrocyte Membrane-specific Antigens common to Several Species of Rodentia

THE rare existence of organ-specific antisera transcending species differences was pointed out by Coombs¹. Similar new organ, or “erythrocyte-specific”, antisera reacting selectively to the erythrocytes of several species of Myomorpha are described here. The antisera, “anti-A”, were prepared by the method of Adachi and Furusawa²: adult guinea-pigs were sensitized with the “antigen-A” (the protein portion of dd mouse erythrocyte membrane). The detailed procedure for preparing labelled antibody, the staining method and its specific reactivity to mouse erythrocytic cells have been reported before³. Circulating erythrocytes from various animals were stained with the fluorescein isothiocyanate-labelled “anti-A”. Smeared cell preparations were fixed with acetone at ? 20° C for 20 min and incubated with the labelled antisera, which had been thoroughly absorbed with the emulsion of mouse liver and kidney to remove the cross-reacting antibodies, at 37° C for 90 min. A light microscope with a darkfield condenser was used for the fluorescent observations using Orsen's interference filter system. Clear fluorescent reactions, though of varied intensities, were observed with those animals classified as Myomorpha, that is, mouse, rat, golden hamster and Chinese hamster (Table 1, Fig. 1). Mammalian erythrocytes other than those of the guinea-pig used as the sensitized animal showed faint fluorescences. Nucleated erythrocytes of the chick, quail, turtle, toad and goldfish were negative to the reaction. The erythrocyte-specific reactivity of the labelled antisera was examined with the liver, kidney and thymus cells of the animals whose erythrocytes displayed intense fluorescences; they showed, however, no fluorescence.     

Maitotoxin, A Presumed Calcium Channel Activator, Induces the Acrosome Reaction in Mussel Spermatozoa

Maitotoxin, a presumed activator of the voltage-sensitive calcium channel, induced the acrosome reaction in the mussel, Mytilus edulis at physiological pH and in the starfish, Asterias amurensis at pH 9.5. The induction of acrosome reaction by maitotoxin depended upon external Ca²⁺ and was inhibited by two types of calcium channel blockers; verapamil and diltiazem. These results suggest that the activation of the voltage-sensitive calcium channel takes an important part in the initiation of acrosome reaction in Mytilus and other animals.     

Properties of Chlorella cells grown under various photo-heterotrophic conditions

1. Chlorella ellipsoidea was cultured under photo-heterotrophicconditions. Cells showed most favorable growth when 0.2% glucosewas added at the start to the inorganic nutrient medium. 2. Treatment with a mixed solvent, methanol/hexane: 4/3, gavehighly decolorized cells. Molasses alone and pyruvate in combinationwith glucose or with molasses and glucose were effective inobtaining a high yield of chlorophyll and carotenoid pigments. 3. The nitrogen content was lower in bleached cells than innormal cells. Protein yield obtained by the urea soaking methodwas higher with bleached cells than with normal cells. 4. Electron microscopic studies revealed that the outer partof normal cells consists of two layers, an electron-dense innerlayer and an electron-lucent outer layer, whereas, that of bleachedcells consists of only an electron-dense layer. (Received August 28, 1969; )     

Ultrastructural Study of Asters Induced by Microinjection with Sperm Centriolar Fraction in Sea Urchin Eggs

Multiple asters can be artificially induced in sea urchin fertilized eggs by the microinjection of the centriolar fraction of sperm homogenate. Investigation was continued by the electron microscopy to determine whether the multi-aster formation was due to the centrioles or the contaminants in the injected sperm fraction. Thirty three asters in 3 operated eggs were thoroughly examined, and we confirmed that the presence of centrioles in the central region of 26 asters. We considered that the rest of them might contained the centrioles in the sections lost during the preparation procedures. Fragmented axoneme, the plug of electron dense material, and the centriolar fossa, which were usually accompanied with the isolated centrioles, disappeared from the centrioles in these multiple asters. However, electron dense, amorphous materials were formed associating with the triplet blades and distributed around the centrioles. Many astral microtubules were terminated in these pericentriolar materials. Results obtained suggest that, although the pericentriolar material is acting as the microtubule organizing center, all multiple asters, except those derived from fertilization (2 asters per egg), are most likely induced by the injected centrioles and not by the contaminants.     

Aster Formation in Sea Urchin Eggs Induced by the injection of Enzyme-Treated Sperm Centrioles

To determine the responsible components of isolated sperm centrioles for the aster induction in sea urchin eggs, the sperm centriolar fraction was treated with various enzymes and was injected into the unfertilized eggs, then the aster formation in first division was observed after fertilization.
Treatment with 1 μg/ml or higher concentration of trypsin inhibited the centriolar activity for aster induction, whereas the treatment with 50 μg/ml of DNase 1, 80 μg/ml of RNase A, 40 μg/ml of RNase T1, or 0.1 μg/ml of trypsin had no inhibitory effect to induce asters. Injection of 0.5 μg/ml of RNase A or 1 mUg/ml of RNase T1 into the egg caused the detention of mitosis at the streak stage. To examine the temperature effect for aster induction, the centriolar fraction was pre-treated with boiling temperature, and it was found that the fraction became incapable to induce any aster.
Results obtained suggest that the effective components of the sperm centriolar fraction to induce asters in the fertilized sea urchin eggs are the proteins but not the nucleic acids. The aster inducing activity is destroyed by heat treatment.