Inhibitory Effects of Diltiazem and Verapamil, Calcium Antagonists, on Fertilization-Induced Increase in the Phosphorylase Reaction in Echiuroid Eggs

The calcium antagonists diltiazem and verapamil at 100 μM caused considerable inhibition of the glycolysis system in recently fertilized eggs of the echiuroid, Urechis unicinctus . The levels of glycolytic intermediates in eggs were found to be higher 5 min after insemination than before fertilization while the levels of adenine nucleotides and inorganic phosphate were almost the same before and after fertilization. Addition of diltiazem or verapamil 30 sec after insemination did not inhibit fertilization, but resulted in maintenance of as low levels of glycolytic intermediates as in unfertilized eggs. The apparent mass action ratio in the phosphorylase step, calculated from the levles of glucose-1-phosphate and inorganic phosphate was normally higher in fertilized eggs than in unfertilized eggs, but was maintained at as low a level as in unfertilized eggs by adding these compounds 30 sec after insemination. Phosphorylase a activity also normally increased after insemination, but was maintained at a low level in fertilized eggs by adding these compounds. These compounds also inhibited the increased ⁴⁵Ca²⁺ uptake normally observed after fertilization. These results suggest that after fertilization, the Ca²⁺ level increases associated with fertilization-induced Ca²⁺ influx and that this stimulates Ca²⁺ dependent protein kinase to phosphorylate phosphorylase b , resulting in an increased rate of the phosphorylase reaction.     

VII. Decrease in the Rate of Respiration in the Spermatozoa of the Sea Urchin, Hemicentrotus Pulcherrimus, Caused by Long Chain Fatty Acyl-CoA-induced Inhibition of the Movement

Spermatozoa of the sea urchin, Hemicentrotus pulcherrimus , showed marked decrease in respiration, and arrested movement after interaction with the fixed eggs. Immotile spermatozoa that had reacted with fixed eggs contained higher levels of long chain fatty acyl-CoAs than normal motile spermatozoa. On treatment with carnitine, the immotile spermatozoa became motile again and their intracellular concentrations of long chain fatty acyl-CoAs decreased. On incubation with anti-mycin A or CN⁻ for 20 min, the motility of normal spermatozoa decreased gradually but their long chain fatty acyl-CoA content changed only slightly. The decrease in sperm motility in the latter case was probably due to decrease in the level of ATP, resulting from inhibition of respiration by antimycin A or CN⁻. The motility of spermatozoa extracted with Triton X-100 was restored by ATP and their movement was inhibited by long chain fatty acyl-CoAs, such as myristoly CoA and palmitoyl-CoA, but was not by short chain fatty acyl-CoAs, such as acetyl-CoA, propionyl CoA and butyryl-CoA. Na-palmitate, Na-myristate and CoA did not inhibit the reactivation of extracted spermatozoa by ATP.     

Activation of Sea Urchin Eggs by Halothane and Its Inhibition by Dantrolene

Eggs of the sea urchin, Hemicentrotus pulcherrimus , were stimulated by halothane, known to induce Ca²⁺ release from sarcosome, to cause fertilization membrane formation in normal and Ca²⁺ free artificial sea water. In the absence of external Ca²⁺, halothane-induced formation of fertilization membrane was inhibited by dantrolene, an inhibitor of Ca²⁺ release from sarcosome, but was not blocked by nifedipine, a Ca²⁺ antagonist specific to Ca²⁺ channels in plasma membrane. Ca²⁺ release from sedimentable fraction isolated from eggs was induced by halothane and was inhibited by dantrolene, but was not blocked by nifedipine. In normal artificial sea water, halothane-caused egg activation was not inhibited either by dantrolene or by nifedipine, but was blocked in the presence of both compounds. ⁴⁵Ca²⁺ influx was substantially stimulated by halothane in eggs exposed to ⁴⁵CaCl₂. Halothane-induced ⁴⁵Ca²⁺ influx into eggs was inhibited by nifedipine but was not blocked by dantrolene. When Ca²⁺ release from intracellular organellae is blocked, Ca²⁺ transport through Ca²⁺ channels in plasma membrane probably acts as a "fail-safe" system to induce an increase in cytosolic Ca²⁺ level, resulting in egg activation.     

Phylogeny of Regulatory Regions of Vertebrate Tyrosinase Genes

Highly homologous DNA elements were found to be shared by the upstream regions of the mouse tyrosinase and tyrosinase related protein (TRP-1) genes. Several nuclear proteins were shown to bind to both of these upstream regions. Shared homologous DNA elements were also found in the 5’ flanking sequences of Japanese quail and snapping turtle tyrosinase genes. Shared homologous nucleotide sequences were found to be scattered like an archipelago in the 5’ upstream regions of mouse and human tyrosinase genes. Comparisons between Japanese quail and snapping turtle tyrosinase genes gave similar results. On the contrary, mammalian (mouse and human) and nonmammalian (quail and snapping turtle) tyrosinase genes did not show significant homology in their 5’ upstream regions. In contrast, coding sequences in the first exons of vertebrate tyrosinase genes and their deduced amino acid sequences were found to be highly conserved except for their putative leader sequence-coding regions.     

Exogut Formation by the Treatment of Sea Urchin Embryos with Ascorbate and α-Ketoglutarate

In sea urchin embryos at the stages from hatch out to the pluteus stage, [¹⁴C]proline incorporation into hot trichloroacetic acid TCA-extractable proteins occurred during an exposure to [¹⁴C]proline for 3 hrs at 20°C. The rate of [¹⁴C]proline incorporation into hot TCA-extractable proteins was higher in gastrulae and plutei than in blastulae. Percentage of [¹⁴C]hydroxyproline residue to whole radioactivity of the hot TCA-extractable proteins was quite low at the blastula stage and increased exponentially during futher development. Production of [¹⁴C]hydroxyproline residue at the blastula stage, as well as at the later stages, was stimulated by ascorbate and α-ketoglutarate, activators of protocollagen proline hydroxylase, and inhibited by α, α'-dipyridyl, an inhibitor of this enzyme. It is also probable that the enzyme in the embryos is not fully activated because of low amounts of activating substances. These suggest that blastulae,…, also have a potency of protocollagen hydroxylation. Blastula kept in sea water containing ascorbateand α-ketoglutarate became undeveloped embryo with large exogut. Gastrula developed normally to pluteus even in the presence of these compounds. The embryos, kept in sea water containing these compounds from fertilization to hatch out, also developed normally. Exogut formation in the embryos treated by these compounds, as well as normal archenteron formation, was inhibited by α, α'-dipyridyl.     

EFFECT OF PROLACTIN ON THE TADPOLE TAIL FIN. I. STIMULATORY EFFECT OF PROLACTIN ON THE COLLAGEN SYNTHESIS OF THE TADPOLE TAIL FIN

Bovine prolactin stimulated the growth of connective tissues both in the tail fins and in other regions of the tadpole tail. Correlated with the morphological effect of the hormone on the tadpole tails, protein synthesis in tail fins was promoted about 2 times by prolactin. Experiments performed to determine the kind of protein, the synthesis of which was stimulated by prolactin, revealed that the hormone specifically enhanced collagen synthesis about 40 folds as compared to untreated animals.     

SOME OBSERVATIONS OF ABNORMAL EMBRYOS INDUCED BY SHORT-PERIOD TREATMENT WITH CHLORAMPHENICOL DURING EARLY DEVELOPMENT OF SEA URCHIN

Sea urchin embryos, which were treated with 5 × 10⁻³ M chloramphenicol for 1 to 4 hr at certain stages before hatching, developed to several types of abnormal embryos. No significant effect on the shape of the embryo was observed when the concentrations of chloramphenicol used in the short-period treatment were lower than 2 × 10⁻³ M. Embryos up to the 2-cell stage, treated with 5 × 10⁻³ M chloramphenicol for a short period, became small blastulae filled with mesenchyme-like cells (type A). A similar effect of puromycin (2 μg/ml) was also observed at this stage. When the chloramphenicol treatment (for 1 to 4 hr) was applied at 8 ∽ 32-cell stages, vegetalized larvae were produced (type B). Embryos treated with chloramphenicol at 7 hr after insemination at 20°C, developed to another type of abnormal larva different from the previous types (type C). A concentration of puromycin (2 μg/ml) which inhibited protein synthesis to the same degree as 5 × 10⁻³ M chloramphenicol, induced only type A. Between these chloramphenicol-sensitive stages, there were chloramphenicol-insensitive stages for forming abnormal embryos.     

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).     

PREVENTION BY HYDROXYUREA OF VEGETALIZATION OF SEA URCHIN LARVAE INDUCED BY cAMP PHOSPHODIESTERASE INHIBITORS

During 3-hr treatment of the morulae of sea urchin with cAMP phosphodiesterase (PDE) inhibitors, which produce vegetalized larvae, incorporation of ³H-thymidine into DNA occurs at almost the same rate as in control embryos. DNase I digests the newly synthesized DNA in chromatin isolated from morulae treated with PDE-inhibitor (caffeine) faster than that isoloated from normal morulae whereas it dogests DNA isolated from chromatin in caffeine treated embryos at almost the same rate as that in normal embryos. Hydroxyurea, an inhibitor of nucleside diphosphate reductase, prevents the vegetalizing effect of PDE-inhibitor on the development of sea urchin embrys.