Eigoro Tazawa

d-Galactoside-Specific Lectins from the Body Wall of an Echiuroid (Urechis unicinctus) and Two Annelids (Neanthes japonica and Marphysa sanguinea)

Lectins recognizing D-galactosides were purified from the body wall of an echiuroid; Urechis unicinctus and two annelids; Neanthes japonica and Marphysa sanguinea, with single step lactosyl-agarose affinity column chromatography. SDS-PAGE under reduced and non-reduced conditions showed that U. unicinctus lectin had a major (36 kDa) and two minor (40 and 14 kDa) proteins, and that N. japonica lectin and M. sanguinea lectin had single 33 and 35 kDa proteins, respectively. Lectins were solubilized in the presence of lactose from tissues, and all polypeptides were shown to have sugar binding activity. The antisera raised against U. unicinctus lectin and N. japonica lectin crossreacted with each other but did not crossreact with bull frog (Rana catesbeiana) egg galectin-1 or a D-galactoside-specific lectin purified from sea urchin (Anthocidaris crassispina) eggs. These echiuroid and annelid lectins are immunologically similar, but distinct from members of the vertebrate galectin family.

Glycolysis in the eggs of the echiuroid, Urechis unicinctus and the oyster, Crassostrea gigas: Rate-limiting steps and activation at fertilization

Abstract The content of glycolytic intermediates and of adenine nucleotides was measured in eggs of the echiuroid, Urechis unicinctus and the oyster, Crassostrea gigas , before and after fertilization. On the whole, the profile of the change in each glycolytic intermediate in Urechis eggs upon fertilization was found to be essentially similar to that in oyster eggs. Calculation of the mass action ratio for each glycolytic step from the amounts of glycolytic intermediates determined suggests that there are at least three limiting enzymes in the glycolysis system in unfertilized and fertilized eggs of each species examined. Phosphorylase (EC 2.4.1.1), phosphofructokinase (EC 2.7.1.11), and pyruvate kinase (EC 2.7.1.40) may be rate-limiting enzymes for the glycolysis system in Urechis eggs as well as in oyster eggs. These enzymes are thought to be activated upon fertilization, though even the reactions of the enzymes in fertilized eggs do not reach a state of equilibrium. In eggs of Urechis and oyster, phosphorylase is the first enzyme to be activated following fertilization. In Urechis eggs, pyruvate kinase is activated after the instant increase in the phosphorylase activity upon fertilization, followed by phosphofructokinase activation. In oyster eggs, however, pyruvate kinase and phosphofructokinase seem to be stimulated simultaneously, subsequent to phosphorylase activation upon fertilization. The mechanism controlling phosphorylase and pyruvate kinase activity is unknown, but the phosphofructokinase activity in both species may be regulated by the intracellular concentration of adenine nucleotides, since the enzyme activity is enhanced along with a decline in the phosphate potential in the eggs of both Urechis and of oyster.

Does the low respiratory rate in unfertilized eggs result mainly from depression of the redox reaction catalyzed by flavoproteins? Analysis of the respiratory system by light-induced release of CO-mediated inhibition

In unfertilized eggs of the sea urchin, the quite low respiratory rate is enhanced by tetramethyl‐p‐phenylenediamine (TMPD), phenazine methosulfate (PMS) and sperm and this augmentation is completely inhibited by carbon monoxide (CO). Exposure to light releases eggs from this CO‐mediated inhibition. The action spectra for photoreactivation of CO‐inhibited cytochrome c oxidase in isolated mitochondria and CO‐blocked respiration in TMPD‐treated eggs were found to be similar to the absorption spectrum of CO‐bound cytochrome aa3. In PMS‐treated eggs and fertilized eggs, the maximum photoreactivation of CO‐inhibited respiration occurred at a light fluence rate higher than that for maximum photoreactivation of CO‐inhibited respiration in TMPD‐treated eggs, with peaks at the same wavelengths as those in the absorption spectrum of reduced cytochrome b. A similar phenomenon was seen for NADH cytochrome c reductase in mitochondria. Thus, cytochrome c oxidase and NADH cytochrome c reductase, whose activities are not altered by fertilization, seem to be functional, even in unfertilized eggs. In unfertilized eggs, difference spectra indicated that PMS and sperm augmented cytochrome b reduction and that TMPD accelerated cytochrome c reduction without cytochrome b reduction. Therefore, it is likely that depression of electron transport to cytochrome b, which is augmented by PMS and sperm, is responsible for the low respiratory rate in unfertilized eggs.

Degeneration of Respiratory System in Sea Urchin Spermatozoa during Incubation in Seawater for Long Duration

Abstract Motility and respiration were examined in spermatozoa of the sea urchin Hemicentrotus pulcherrimus after dilution and incubation in seawater at pH 8.2 at 20°C. Almost all spermatozoa were motile during 12 hr of incubation, but their respiratory rate decreased gradually. The acrosome reaction was also induced by egg jelly solution during 12 hr of incubation in seawater. However, the ratio of spontaneous acrosome reacted spermatozoa was quite low during the same period. An intracellular pH (pHi) of spermatozoa was about 7.5 just after dilution in seawater and was almost constant during 12 hr of incubation. Upon dilution and incubation in seawater, activity of NADH-cytochrome c reductase decreased in propotion to the decrease in the respiration in spermatozoa, whereas cytochrome c oxidase activity was hardly changed. These suggest that the degeneration of respiratory system during 12 hr of incubation in seawater is due to the decrease in the NADH-cytochrome c reductase activity. In energy production system, phosphatidylcholine as a preferred substrate was efficiently hydrolyzed during 4 hr of incubation and then the activity of the energy metabolism decreased gradually. Beyond 12 hr incubation in seawater, the number of immotile spermatozoa increased and respiratory rate declined rapidly. Also, the percentage of the acrosome reaction induced by the egg jelly solution decreased. These are probably due to the increase in the number of dead spermatozoa after 12 hr of incubation in seawater. It is thus concluded that the life-span of H. pulcherrimus spermatozoa is about 12 hr after dilution in seawater.

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.

Activation of co-insensitive respiration in echiuroid eggs by light irradiation at various wavelengths

Abstract 1. 1. The respiratory rate of echiuroid eggs in the presence of CO was slightly lower than in its absence and became markedly high by light irradiation at all examined wavelengths between 350 and 650 nm. 2. 2. The action spectrum for photo-activation of respiration in the presence of CO exhibited peaks of activating effect of light at 430, 530 and 570 nm. 3. 3. The similarity of this action spectrum to the absorption spectrum of reduced cytochrome b suggests that photo-activation of CO-insensitive respiration results from conversion of inactive cytochrome b to active form by its photon energy absorption.

Photo-activation of respiration in the presence of CO in sperm of several marine invertebrates

Abstract 1. 1. The light irradiation at examined wavelengths between 360 and 630 nm enhanced the respiratory rate in the presence of CO in sperm, as well as eggs, of the sea urchins, Anthocidaris crassispina and Hemicentrotus pulcherrimus , the starfish, Asterina pectinifera , and the echiuroid, Urechis unicinctus . 2. 2. The maximum peaks of stimulating effects of light irradiation on CO-insensitive respiration were found at wavelengths of 430, 530 and 570 nm in sperm of these species. 3. 3. The respiration in the presence of CO was insensitive to light irradiation in sperm and eggs of the oyster, Crassostrea gigas and the tunicate, Ciona intestinaris .

Vegetalization Induced by Procaine and Tetracaine in Sea Urchin Embryos

Vegetalization of sea urchin embryos was induced by the treatment with procaine and tetracaine, inhibitors of Ca2+mobilization, for 3 hr starting 3–5 hr after insemination at 20°C. The treatment starting 7 hr after insemination sometimes produced similar type of vegetalized embryos. The pulse treatment starting at the other stages hardly yielded vegetalized embryos. The stages at which these compounds were effective to produce vegetalized embryos were almost the same to those for Li+to make embryos vegetalized. On the basis of known inhibitory effects of tetracaine, procaine and Li+on Ca2+mobilization, we postulate that Ca2+dependent reactions participate in the process of cell determination at these stages. Inhibitory effects of procaine, tetracaine and Li+on Ca2+dependent induction of fertilization membrane formation, found in the present study, indicate that these compounds block Ca2+mobilization in sea urchin eggs.

Fertilization-induced change in the respiratory rate in eggs of several marine invertebrates

Abstract 1. 1. Fertilization enhances the respiratory rate in eggs of sea urchin and tunicate but hardly increases it in oyster, mussel, echiuroid, polychaete and starfish. In all examined species, fertilization increased the rate under infinite stimulation by 2,4-dinitrophenol. 2. 2. In these eggs, the responses of respiration to phenazine methosulfate and 2,4-dinitrophenol suggest that fertilization releases blockages of mitochondrial respiration and its coupling to oxidative phosphorylation. 3. 3. In the former two species, the blockage of respiration is quite strong in unfertilized eggs and hence, its release by fertilization probably becomes apparent in spite of intensified ADP control by releasing the blockage in coupling to phosphorylation.

Respiration in Eggs of the Echiuroid, Urechis unicinctus, Before and After Fertilization

In eggs of the echiuroid Urechis unicinctus the respiration rate, which is not altered by fertilization, is inhibited by rotenone, antimycin A and cyanide. The respiration in echiuroid eggs is probably mediated by the mitochondrial respiratory chain. In fertilized eggs, the respiration was inhibited by oligomycin and stimulated by the uncouplers of oxidative phosphorylation 2,4‐dinitrophenol and carbonylcyanide p‐trifluoromethoxyphenylhydrazone, whereas respiration in unfertilized eggs was insensitive to these compounds. Insemination increased the respiratory rate in eggs in the presence of uncouplers and reduced it in the presence of oligomycin. These findings suggest that the capacity of electron transport in mitochondira is elevated by fertilization but becomes latent on fertilization‐induced coupling of respiration with oxidative phosphorylation. Strong stimulation of the respiration in unfertilized eggs was induced by dichlorophenol indophenol, phenazine methosulfate and tetramethyl p‐phenylenediamine, suggesting possible sites at which electron transport is regulated in unfertilized eggs. The resulting stimulation of respiration in unfertilized eggs was insensitive to uncouplers and oligomycin, but became sensitive to them after fertilization simultaneously with considerable decrease in its rate. Fertilization‐induced coupling of the respiration seemed to reduce the respiratory rate enhanced artificially by these redox compounds.