Kouichi Asami

Ca2+ uptake H+ ejection and respiration in sea urchin eggs on fertilization.

Abstract Changes in Ca2+ uptake, H+ ejection and respiration were examined immediately after fertilization in two species of sea urchin eggs. These three phenomena occurred simultaneously with a small lag and were followed by considerably high rates of each process. H+ ejection occurred parallel with the uptake of Ca2+ as shown in the cessation of processes by addition of GEDTA. The respiration of egg homogenates after fertilization was stimulated by the addition of Ca2+. The possible role of Ca2+ at the onset of the biochemical process after fertilization is discussed.

Glycolysis of sea urchin eggs: Rate-limiting steps and activation at fertilization

The amounts of glycolytic intermediates and adenine nucleotides in unfertilized Anthocidaris crassispina eggs and in fertilized eggs or embryos were measured. The determinations on unfertilized and fertilized (30 min) eggs of Pseudocentrotus depressus showed the same results. Calculation of both mass action ratios and free energy changes for each enzymatic step of glycolysis showed that reactions catalysed by α-glucan phosphorylase (EC 2.4.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) were rate-limiting steps of glycolysis in both unfertilized and fertilized eggs. It also suggested that these three key or rate-limiting enzymes were activated by fertilization. Phosphorylase is activated at fertilization as is also pyruvate kinase. Activation of phosphorylase is also shown by the measurement of the activity in homogenate. Phosphofructokinase showed no increase in activity until 20 min after fertilization, the increase then being closely correlated with a decline in phosphate potential. On the basis of their mass action ratios, none of these rate-limiting enzymes appears to have reached a state of equilibrium by hatching (20 h). The temporal discontinuities in the activation pattern of these three enzymes suggests that no single control mechanism can be operative during the first hour following fertilization.

Effect of chronic HTO. beta. or /sup 60/Co. gamma. radiation on preimplantation mouse development in vitro

Response of pronuclear, early 2-cell, and late 2-cell mouse embryos to chronic HTO β and60 Co γ irradiation was investigated. The pronuclear embryos fertilized in vitro and 2-cell stage embryos of ...

Distribution of some enzymes concerning carbohydrate metabolism in sea urchin eggs.

Abstract As an aid to the elucidation of the mechanism of activation of glycolysis upon fertilization, the activity and the distribution of the enzymes concerned were measured in unfertilized and fertilized eggs of Hemicentrotus pulcherrimus and Pseudocentrotus depressus . The enzymes investigated were phosphorylase, exo-1,4-α-glucosidase, hexokinase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, 6-phosphofructokinase, hexosediphosphatase, fructose-bisphosphate aldolase, pyruvate kinase, and lactate dehydrogenase. Phosphorylase and pyruvate kinase were the enzymes which were activated upon fertilization. Glucose-6-phosphate dehydrogenase and a part of aldolase changed their distribution from the particulate to the soluble fraction upon fertilization. Advantages of enzyme activation over changes in enzyme distribution upon fertilization were discussed as a mechanism for the fertilization-induced activation of glycolysis.

Activation of phosphorylase in sea urchin eggs by Ca2+ and cyclic 3′5′-AMP: A possible mechanism of the regulation of its activity at fertilization

Abstract γ-Glucan phosphorylase (EC 2.4.1.1) activity in homogenates of unfertilized and fertilized sea urchin eggs, Pseudocentrotus depressus and Hemicentrotus pulcherrimus , has been studied. The phosphorylase exhibits a pH optimum at 6.4 and occurs in two forms, AMP-independent and AMP-dependent, the latter showing maximum activity in the presence of 10 mM AMP. By as little as 5 min after insemination a significant increase in the total phosphorylase activity of the egg as well as in the AMP-independent form is demonstrable. The highest specific enzyme activity is consistently found in the supernatant fraction of both the fertilized and the unfertilized egg homogenate. Thus, fertilization does not appear to cause activation of the enzyme by stimulating a change from a particulate-bound to a soluble state. The phosphorylase activity was compared after incubation of homogenates with a variety of agents potentially able to alter the enzyme activity. Combination of suitable amount of cyclic 3′5′-AMP (cAMP) and Ca 2+ showed the maximal activating effect on the AMP-independent form of phosphorylase. The fertilization-induced increase of Ca 2+ and of cAMP were discussed as possible activators of phosphorylase, and consequently, of carbohydrate metabolism.

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.

Inhibition by x-irradiation and antimetabolites of dna synthesis without affecting camp elevation in isoproterenol-stimulated mouse parotid gland.

Abstract The concentration of adenosine 3′,5′-cyclic monophosphate (cAMP) was measured in the parotid gland of the mouse after intraperitoneal injection of a beta-adrenergic drug, isoproterenol. A marked elevation of cAMP was observed 10 min after the administration, followed by a return to the control level within 40 min. A small peak was found around 14 h, onset of the stimulated DNA synthesis being observed at 20 h. A close relationship was found between the cAMP level at 10 min and the rate of DNA synthesis at 24 h in animals given different doses of the drug. However, DNA synthesis could not be induced by adrenalin in spite of a significant increase in the cAMP concentration. Furthermore, X-irradiation or certain metabolic inhibitors (actinomycin D and cycloheximide), administered prior to isoproterenol, completely inhibited the stimulated DNA synthesis without affecting the cAMP elevation at 10 min. It is concluded that the critical step in the initiation of stimulated DNA synthesis may be located at a period later than the initial cAMP elevation.

Synthesis and phosphorylation of histone H1 and high mobility group proteins in the regenerating rat liver after X irradiation

A rat was irradiated to the upper abdomen including the liver and then partially hepatectomized. The subsequent synthesis and phosphorylation of histone H1 and nonhistone chromosomal high mobility group (HMG) proteins were investigated. Incorporation of [3H]lysine into histone H1 was increased and reached its peak at 27 h after hepatectomy, and 14 Gy of X rays inhibited the increase. Increase in the incorporation of [3H]lysine into HMG (1 + 2), 14, and 17 which occurred around 27 h after hepatectomy was not inhibited by 14 Gy irradiation. Phosphorylation of histone H1, measured with 32Pi incorporation in vivo, was maximal between 21 and 24 h, and it was inhibited by 4.8 Gy of X rays and delayed with 1.9 Gy. Phosphorylation of HMG 14, which was the only HMG protein phosphorylated under present conditions, was not affected by X irradiation. The [3H]thymidine incorporation into nuclear DNA started increasing at 21 h and reached its maximum at 27 h after hepatectomy. X irradiation with 4.8 Gy inhibited the incorporation, and 1.9 Gy lowered it.

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.

Induction of Fertilization Membrane Formation and Cyanide‐insensitive Respiration in Sea Urchin Eggs by the Treatment with Dimethylsulfoxide Followed by an Incubation in an Ice Bath

In unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus, fertilization membrane formation was induced by an incubation with dimethylsulfoxide (DMSO) for several min at 20°c followed by another incubation in an ice bath. The number of eggs with fertilization membrane, thus obtained, increased in relation to the concentration of DMSO between 1 and 3% (v/v) and was higher than 75% at concentrations above 3%. Fertilization membrane formation by this treatment occurred in Ca2+ free‐ or Ca2+, Mg2+ free‐ artificial sea water containing EGTA (50 mM) and was inhibited by verapamil. In the presence of DMSO, the membrane formation was also induced by 2, 4‐dinitrophenol or cyanide in considerable number of eggs at 20°c. Eggs remained fertilizable, even when they were kept with DMSO for 1 hr at 20°c. DMSO slightly enhanced respiratory rate in unfertilized eggs and substantially reduced it in fertilized eggs. DMSO‐treated eggs exhibited cyanide‐insensitive respiratory burst following chilling in an ice bath or by adding DNP or cyanide, in a similar manner to the burst induced by sperm.