Keiko Mitsunaga

Isolation of cDNAs for LCE and HCE, two constituent proteases of the hatching enzyme of Oryzias latipes, and concurrent expression of their mRNAs during development.

The hatching enzyme of medaka consists of two types of proteases (HCE, LCE). cDNA clones for LCE and HCE were isolated from a lambda gt11 cDNA library constructed with poly(A)+ RNA of Day 3 embryos. LCE cDNA is 936 bp long and contains an 813-bp open reading frame encoding a preproenzyme with a 20-amino-acid signal sequence, a 51-amino-acid propeptide, and a 200-amino-acid mature enzyme. For HCE, two distinct cDNAs (HCE21, HCE23) having nucleotide sequences with 92.8% similarity were obtained. These cDNAs contain open reading frames encoding preproenzymes of 279 and 270 amino acids, respectively. The mature enzyme forms of both consist of 200 amino acids, the similarity between them being 95.5%. On Northern blotting analysis, the transcripts of LCE and HCE genes were first detected coincidentally in Day 2 embryos shortly before the production of LCE and HCE, accumulated thereafter in parallel, and dramatically decreased after hatching. The amino acid sequence, the HExxH motif, which is known to constitute an active site in some Zn proteases, is also found in LCE and HCE. However, the sequence analyses strongly suggest that both the enzymes belong to the astacin (protease) family, being distinct from sea urchin hatching enzyme, which is reportedly similar to collagenase.

Carbonic anhydrase activity in developing sea urchin embryos with special reference to calcification of spicules

Eggs and embryos of the sea urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus did not exhibit significant changes in carbonic anhydrase activity during early development. Acetazolamide inhibited enzyme activity in homogenates of embryos and inhibited the formation of calcified spicules in a culture of micromeres at concentrations between 40 and 100 microM. Acetazolamide allowed intact embryos to develop to quasi-normal plutei but inhibited calcium deposition in the spicules. It is suggested that carbonic anhydrase contributes to CaCO3 deposition in the spicule.

Probable Role of Allylisothiocyanate‐Sensitive H+, K+‐ATPase in Spicule Calcification in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

In embryos of the sea urchin, Hemicentrotus pulcherrimus, as well as in cultured cells derived from isolated micromeres, spicule formation was inhibited by allylisothiocyanate, an inhibitor of H+, K+‐ATPase, at above 0.5 μM and was almost completely blocked at above 10 μM. Amiloride, an inhibitor of Na+, H+ antiporter, at above 100 μM exerted only slight inhibitory effect, if any, on spicule formation. Intravesicular acidification, determined using [dimethylamine‐14C]‐aminopyrine as a pH probe, was observed in the presence of ATP and 200 mM KCl in microsome fraction obtained from embryos at the post gastrula stage, at which embryos underwent spicule calcification. Intravesicular acidification and K+‐dependent ATPase activity were almost completely inhibited by allylisothiocyanate at 10 μM. Allylisothiocyanate‐sensitive ATPase activity was found mainly in the mesenchyme cells with spicules isolated from prisms. H+, K+‐ATPase, an H+ pump, probably mediates H+ release to accelerate CaCO3 deposition from Ca2+, CO2 and H2O in the primary mesenchyme cells. Intravesicular acidification was stimulated by valinomycin at the late gastrula and the prism stages but not at the pluteus stage. K+ permeability probably increases after the prism stage to activate H+ release.

Stage specific effects of Zn2+ on sea urchin embryogenesis

The treatment of sea urchin embryos by Zn2+ followed by culture with Zn2+‐specific chelators such as ethylenediamine‐N, N′‐diacetic acid and N‐hydroxyethylethylenediamine‐N, N′, N′‐triacetic acid, was performed at various developmental stages to find out specific stages for Zn2+ to induce abnormal differentiation. The treatment with 1 mM ZnSO4 at 20°C during a period including two spans of development between 0 and 8 hr and between 14 and 16 hr post fertilization yielded permanent blastulae. Zn2+‐treatment during the former span produced abnormal prisms and plutei with small archenteron. The treatment for a period including only the latter span failed to produce abnormal ones. Zn2+‐treatment during a period including the gastrula stage also produced abnormal spherical embryos. Without the culture with these chelators, abnormal embryos were produced by Zn2+‐treatment performed at any stages before gastrulation. A high zinc amount in the embryos just after the treatment became as low as in normal embryos soon after the culture with these chelators and was maintained during the culture without them. These results indicate that zinc retention occurs in the Zn2+‐treated embryos and causes abnormal differentiation when the treated embryos develop in normal sea water through the Zn2+‐specific periods of development.

Probable contribution of protein phosphorylation by protein kinase C to spicule formation in sea urchin embryos

The formation of spicules and development of pluteus arms in sea urchin embryos were strongly blocked by H‐7 (1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine dihydrochloride) but were not affected by HA1004 (N‐(2‐guanidinoethyl)‐5‐isoquinolinesulfonamide hydrochloride). Archenteron formation occurred normally in the presence of these compounds. Late gastrulae (28 hr after fertilization) were exposed to 32Pi for 30 min at 20°C, and then dissociated and their primary mesenchyme cells with spicules, embryo‐wall cells and archenteron cells were separated. Then, the radioactivities in the protein fractions of these separated cells were measured. Results showed that culture of embryos with H‐7 strongly inhibited 32p incorporation into proteins in primary mesenchyme cells but caused little inhibition of its incorporations in embryo‐wall cells and archenteron cells. The effective concentrations of H‐7 for inhibition of 32p incorporation were within the range that blocked spicule formation and growth of pluteus arms in embryos. HA1004 only slightly inhibited 32p incorporation into protein in mesenchyme cells, embryo‐wall cells and archenteron cells of embryos exposed to 32Pi. Protein kinase C activity was detectable only in isolated primary mesenchyme cells associated with spicule structures. These suggest that phosphorylation of proteins by protein kinase C contributes to the formation of spicule structures.

Inhibitory Effect of Omeprazole, a Specific Inhibitor of H+, K+‐ATPase, on Spicule Formation in Sea Urchin Embryos and in Cultured Micromere‐Derived Cells.

Embryos kept with omeprazole, a specific H+, K+‐ATPase inhibitor, in a period of development between the mesenchyme blastula and the pluteus corresponding stage became abnormal plutei having quite small spicules, somewhat poor pluteus arms and apparently normal archenterons. In micro‐mere‐derived cells, kept with omeprazole at pH 8.2 in a period between 15 and 40 hr of culture at 20°C, omeprazole strongly inhibited spicule formation but did not block the outgrowth of pseudopodial cables, in which spicule rods were to be formed. These indicate that omeprazole probably exerts no obvious inhibitory effects other than spicule rods formation. Omeprazole‐sensitive H+, K+‐ATPase, an H+pump, seems to be indispensable for CaCO3 deposition (formation of spicule rod) in these spicule forming cells. H+, produced in overall reaction for CaCO3 formation: Ca2++ CO2+H2O°CaCO3+2H+, is probably released from the cells by this H+pump and hence, this reaction tends to go to CaCO3 production to form spicule rods. Omeprazole, known to become effective following its conversion to a specific inhibitor of H+, K+‐ATPase at acidic pH, is able to inhibit formation of spicule rod at alkaline pH in sea water. This is probably due to an acidification of sea water near the cell surface by H+ejection in H+, K+‐ATPase reaction.

Changes in the Activities of H+, K+‐ATPase and Na+, K+‐ATPase in Cultured Cells Derived from Micromeres of Sea Urchin Embryos with Special Reference to Their Roles in Spicule Rod Formation

In cultured cells derived from micromeres isolated at the 16‐cell stage of sea urchin embryos, the activity of H+, K+‐ATPase became detectable after 15 hr of culture, when the cells started to form spicules, and then increased reaching a plateau from 25 hr of culture. The Na+, K+‐ATPase activity of isolated micromeres increased to a maximum at 20 hr of culture and thereafter decreased gradually. Allylisothiocyanate, an inhibitor of H+, K+‐ATPase, caused a decrease in intracellular pH (pHi) accompanied by blockage of 45Ca deposition in spicule rods in spicule‐forming cells at 30 hr of culture. Ouabain and amiloride had scarcely any effect on the pHi or 45, deposition. In cultured cells exposed to nifedipine, which blocked 45Ca deposition in spicule rods, allylisothiocyanate did not cause any decrease in pHi. These results show that H+, which is generated in the overall reaction to produce CaCO3 from Ca2+ and HCO3−, is probably released from the cells mainly in the reaction catalyzed by H+, K+‐ATPase to maintain successive production of CaCO3.

Does Protein Phosphorylation by Protein Kinase C Support Pseudopodial Cable Growth in Cultured Micromere‐Derived Cells of the Sea Urchin, Hemicentrotus pulcherrimus?

In cultured cells derived from micromeres, H‐7 strongly inhibited the outgrowth of pseudopodial cables and the formation of spicule rods at concentrations around the Ki values for protein kinases. HA1004 did not inhibit the cable growth and spicule rod formation in these cells at higher concentrations than the Ki values for cyclic nucleotide‐dependent protein kinases. Pseudopodial cable growth was also inhibited by H‐7 in furosemide‐treated cells which were able to undergo normal growth of the cables without the formation of spicule rods. Protein phosphorylation, measured by 32P incorporation into proteins in the cells exposed to 32Pi, was inhibited by H‐7 at the concentrations for the blockage of the cable growth but was hardly blocked by HA1004. The cable growth and protein phosphorylation were activated by phorbol 12‐myristate 13‐acetate. The activity of Ca2+, phospholipid‐dependent protein kinase (protein kinase C), which was inhibited by H‐7, became appreciably high in micromere‐derived cells at 16 hr of culture at 20°C, at which the outgrowth of pseudopodial cables was going to be initiated and gradually increased keeping pace with the cable growth. These suggest that the outgrowth of the cables is supported by protein phosphorylation catalyzed by protein kinase C.

Development of Sea Urchin Embryos in Artificial Sea Water Containing Br−in Place of Cl−

In artificial sea water in which the Cl−concentration was reduced to less than 10% of that in normal sea water by its replacement with Br−, sea urchin eggs were fertilized and developed into abnormal plutei following almost the same time schedule as in natural sea water. These embryos had poorly developed spicules, short pluteus arms, somewhat jagged embryo‐walls and quasi‐normal archenterons. Similar embryos were obtained in another artificial sea water in which 90% of the Cl−concentration in normal sea water was reduced by Br−and 10% by acetate. In artificial sea water, in which either 90% of the Cl−was replaced by Br−or 10% was replaced by acetate, embryos developed into plutei with quasi‐normal spicules, pluteus arms and archenterons. These findings indicate that deficiency of Cl−results in somewhat abnormal sea urchin embryos. When cells derived from isolated micromeres, were cultured in these Cl−‐deficient artificial sea waters, containing Br−in place of more than 70% of the normal Cl−concentration in sea water, spicule formation was strongly inhibited, but pseudopodial cables were well developed. Thus, external Cl−seems to be necessary for at least normal formation of spicule rods.