Introduction and Expression of Recombinant β-Galactosidase Genes in Cleavage Stage Mouse Embryos

In an attempt to study gene regulation in very early stages of mouse embryogenesis, we injected genes constructed by joining the coding sequence of the bacterial β-galactosidase gene to four different animal gene enhancers/promoters and to poly (A) signals, and examined the gene expression in cleavage stage embryos.
With appropriate injection volumes for each embryonic stage, ranging from 0.2 to 1.3 pl, the majority of the injected embryos underwent at least one further cleavage. Expression of injected genes, which occurred transiently after injection, required the promoter sequences but without much distinction of the source of enhancer/promoter complexes. This result was in a sharp contrast to transfection of mouse cell lines where the recombinant genes were variably expressed reflecting differential enhancer effects.
By injection at the 1-cell stage, expression of injected genes was low while the expression by injection at the 2-cell or later stages was several fold higher, which may correlate with the fact that most zygotic gene expression begins after the 2-cell stage. The low expression at the 1-cell stage was augmented by the conditions causing clea***age arrest such as inhibition of DNA synthesis with aphidicolin.     

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.     

Physiological activity of a viridicatin derivative, 3-(4-phenylcarbostyriloxy) acetic acid

A carboxymethylene derivative (V-OCH₂COOH) of viridicatin (V-OH)promoted the root growth of rice and sesame seedlings. V-OCH₂COOHhad no known hormonal activities, per se, but did have an inhibitoryeffect on IAA and 2,4-D-induced growth of Avena coleoptile sectionsand of carrot root callus. However, inhibition by VOCH2COOHof 2,4-D-induced growth in carrot root callus was to some extentreversed by increasing the concentration of 2,4-D. V-OCH₂C0OHseemed to competitively inhibit IAA-induced elongation of Avenacoleoptile sections. (Received September 14, 1970; )     

Analysis of the Role of Astral Rays in Pronuclear Migration in Sand Dollar Eggs by the Colcemid-UV Method

The formation and migration of the sperm aster, and the migration of male and female pronuclei during fertilization were investigated in the eggs of the sand dollar, Clypeaster japonicus using the Colcemid-UV method. When an egg in Colcemid sea water was irradiated locally with UV light (about 365 nm wavelength) at a limited region containing sperm head, a sperm aster formed in this region, and migrated to the center of the UV-irradiated region during its formation. When the UV-irradiated region was displaced or its shape was changed after the formation of the sperm aster, the aster migrated to the center of the new UV-irradiated region. The direction of the migration of the sperm aster coincided with the direction of the longest astral rays. Direct contact between astral rays and the egg surface was not essential for sperm aster migration. When a region containing both the sperm centrosome and the female pronucleus was irradiated with UV light, the female pronucleus migrated toward the center of the sperm aster after they were connected by astral rays. The migration was suppressed when UV light was shaded over the region between the aster and the female pronucleus. These results suggest that the female pronucleus migrates to the sperm aster by attractive force between them.     

Morphological and anatomical analyses of rheophytic Rhododendron ripense Makino (Ericaceae)

The comparative morphology and anatomy of the leaves of the rheophytic Rhododendron ripense and the closely related inland species Rhododendron macrosepalum were examined. The leaf of R. ripense is thinner than that of R. macrosepalum, with leaf length to width ratios (leaf index) of 2.92 and 1.91, respectively. Moreover, the leaf of R. ripense consists of fewer cells than the leaf of R. macrosepalum, suggesting stenophyllization of R. ripense caused by the decreased number of cells. In addition, leaf thickness and the number of stomata per leaf of R. ripense were significantly greater than those of R. macrosepalum, but the density of the short glandular pilose hairs on the leaf of R. ripense was lower. The observed morphological differences between the two species may be explained by certain aspects of the riparian environment, such as high irradiation and frequent flooding after heavy rainfall, to which R. ripense is exposed.     

Ultrasonic courtship songs of male Asian corn borer moths assist copulation attempts by making the females motionless

Males of the Asian corn borer moth Ostrinia furnacalis (Guenée) produce an ultrasonic courtship song of extremely low‐intensity during copulation attempts. The song has been shown to significantly increase the mating success of the male; however, the mode of action of the sound in courtship remains to be resolved. Behavioural experiments using pairs with deafened females or muted males show that, without the aid of the sound, 63% of males eventually succeed in mating after several copulation attempts, whereas the remainder (37%) make repeated attempts in vain until interrupted by the escape of the female. Because few (2%) males fail to copulate when females hear the courtship song, it is evident that the song has an effect on females, promoting the success of copulation attempts. In support of this view, males produce louder songs if the first copulation attempt fails, suggesting that the males increase their sound levels to achieve successful copulation. It is suggested that the ultrasonic songs of the male render the females motionless, which is the same response as that to ultrasonic bat calls. Because even slight movements by the female can interfere with the attempt of the male to copulate, it is likely that, by making her motionless, the success rate of a single copulation attempt is increased greatly.     

Fusion of Spermatozoa with Embryonic Cells and Somatic Cells in the Sea Urchin

Spermatozoa can enter the separated blastomeres of 8- and 16-cell stage embryos, the cells of blastulae and even somatic cells of the oesophagus wall of an adult sea urchin, under certain conditions. In the presence of egg jelly solution, the rate of entrance of spermatozoa is remarkably increased. In the case of the blastomere of 8-cell stage embryos, characteristic cytoplasmic protrusions are formed at the sites of sperm entry, in succession to the formation of the cytoplasmic bulge. These protrusions elongate until 4 min after insemination, and then they retract gradually. The nucleus of penetrated sperm swells and decondenses to form a pronucleus. In most cases, the pronucleus seems to fuse with the preexisting diploid nucleus of the blastomere. When the dissociated oesophagus cells were inseminated, a certain type of the cells was found to fuse with spermatozoa, although the percentage of fused cells was very low.     

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.