Ikuo Yasumasu

SPECIES-DEPENDENT MIGRATION OF FISH HATCHING GLAND CELLS THAT COMMONLY EXPRESS ASTACIN-LIKE PROTEASES IN COMMON

Two constituent proteases of the hatching enzyme of the medaka (Oryzias latipes), choriolysin H (HCE) and choriolysin L (LCE), belong to the astacin protease family. Astacin family proteases have a consensus amino acid sequence of HExxHxxGFxHExxRxDR motif in their active site region. In addition, HCE and LCE have a consensus sequence, SIMHYGR, in the downstream of the active site. Oligonucleotide primers were constructed that corresponded to the above‐mentioned amino acid sequences and polymerase chain reactions were performed in zebrafish (Brachydanio rerio) and masu salmon (Oncorynchus masou) embryos. Using the amplified fragments as probes, two full‐length cDNA were isolated from each cDNA library of the zebrafish and the masu salmon. The predicted amino acid sequences of the cDNA were similar to that of the medaka enzymes, more similar to HCE than to LCE, and it was conjectured that hatching enzymes of zebrafish and masu salmon also belonged to the astacin protease family. The final location of hatching gland cells in the three fish species: medaka, zebrafish and masu salmon, is different. The hatching gland cells of medaka are finally located in the epithelium of the pharyngeal cavity, those of zebrafish are in the epidermis of the yolk sac, and those of masu salmon are both in the epithelium of the pharyngeal cavity and the lateral epidermis of the head. However, in the present study, it was found that the hatching gland cells of zebrafish and masu salmon originated from the anterior end of the hypoblast, the Polster, as did those of medaka by in situ hybridization. It was clarified, therefore, that such difference in the final location of hatching gland cells among these species resulted from the difference in the migratory route of the hatching gland cells after the Polster region.

Periodic change in the content of adenosine 3′5′-cyclic monophosphate with close relation to the cycle of cleavage in the sea urchin egg

Abstract The content of adenosine 3′5′-cyclic monophosphate (cAMP) in sea urchin eggs, Hemicentrotus pulcherrimus , increased gradually after fertilization to about 10-fold that in unfertilized egg, and decreased rapidly during cytokinesis of the egg to the level found in unfertilized egg. The same profile of the change in cAMP content as found during first cleavage, was also observed during second and third cleavage. The periodic change in cAMP content in the sea urchin egg seems to be repeated with close relation to the cycle of cytokinesis.

Synergistic action of prolactin and androgen on the cloacal glands of the newt

Abstract 1. 1. Injections of prolactin in combination with testosterone propionate (TP) to hypophysectomized or hypophysectomized and castrated newts enhanced the development of tubular cells and secretion in the cloacal gland. 2. 2. Incorporation of 14 C-glucose into the TCA-insoluble materials in the lateral gland was stimulated by prolactin when TP was administered simultaneously. Other pituitary hormones (FSH, LH and GH) did not exhibit such a synergistic action. 3. 3. Synthesis of acid mucopolysaccharides in the lateral gland was stimulated much more by prolactin and TP than prolactin or TP alone.

Inhibitory effect of α-hydrazinoornithine on egg cleavage in sea urchin eggs

Abstract In fertilized sea urchin eggs which are kept in sea water containing α-hydrazinoornithine (αHO) at a concentration above 1 mM from the time of fertilization, cleavage is delayed markedly. The third cleavage is almost completely blocked by 3 mM αHO. Hydrazine, as well as ornithine, exerts no harmful effect on egg cleavage. αHO causes competitive inhibition of ornithine decarboxylase (ODC) in the egg homogenate. Polyamine levels decrease in fertilized eggs treated with αHO. The addition of ornithine (above 3 mM) to an egg culture containing αHO prevents the αHO-induced delay of cleavage. Putrescine (0.2–0.5 mM), which is the product of the reaction catalyzed by ODC, also relieves egg cleavage from the inhibited state. The same effect occurs in the presence of spermidine (0.2–0.5 mM) or spermine (0.1–0.8 mM). Especially, spermine (0.5 mM) completely cancels the inhibitory effect of αHO on egg cleavage. Egg cleavage is delayed only slightly in the presence of each polyamine (above 2 mM).

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.

EFFECT OF TEMPERATURE ON INTERACTION BETWEEN EGGS AND SPERMATOZOA OF SEA URCHIN

Fertilization in the sea urchin, Anthocidaris crassispina, showed marked temperature dependence; high temperatures (15°-30°C) were required for fertilization. In contrast, fertilization in Hemicentrotus pulcherrimus occurred over a wide range of temperatures (0°-30°C). The mechanism of this temperature effect in Anthocidaris was investigated. The number of sperm bound to the egg surface and the rate of the acrosome reaction were markedly reduced by lower temperatures (0°-10°C). Furthermore, an abnormal elongation of the sperm head tip occurred with higher frequency at lower temperatures. In contrast, the egg activation with calcium ionophore A23187 was not prevented at 10°C. The swimming activity measured by distance traveled was also relatively high at 0°C, although the activity increased as the temperature rose. These results strongly suggest that temperature exerts a direct influence on fertilization in Anthocidaris by acting on the acrosome reaction. The increased fertilization rate at higher temperat...

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.

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.

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.

Cyclic change in polyamine concentrations in sea urchin eggs related with cleavage cycle

Abstract Spermidine and spermine are found in unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus . Putrescine becomes detectable and concentrations of spermidine and spermine increase in the eggs upon fertilization. Then, concentrations of these polyamines decrease after respective peaks in polyamine concentrations. The peaks in the concentrations are found at 15 minutes post fertilization for putrescien, at 30 minutes for spermidine and at 30–40 minutes for spermine respectively. Levels of polyamines elevate again and reduce after the 2nd concentration peaks of respective compounds, and then the first cleavage of the eggs takes place. Cyclic change in each polyamine concentration is also observed after the first cleavage, and egg cleavage occurs at decreasing phase of polyamine concentrations.