CHANGES OF THE PRESPORE SPECIFIC STRUCTURE DURING DEDIFFERENTIATION AND CELL TYPE CONVERSION OF A SLIME MOLD CELL

In the slug of the cellular slime mold, Dictyostelium discoideum , are differentiated the anterior prestalk cells and the posterior prespore cells, whose differentiation is characterized by formation of the prespore specific vacuole (PSV). The ultrastructural changes of the PSV were investigated during dedifferentiation of a prespore cell disaggregated from a slug and also during conversion of the cell type, caused by fragmentation of a slug, between the prespore and the prestalk cells.
During the dedifferentiation, the PSV first lost its lining membrane which subsequently congregated, together with the inner filamentous material, to form some electron dense granules. Finally, the vacuole membrane was punctured, and the granules were released into cytoplasm. During conversion of the prespore to the prestalk cell, the PSV was degraded through the same process as in dedifferentiation, but the degradation proceeded much more synchronously in a converting cell. When a prestalk fragment was isolated from a slug, formation of the PSV was detected in no cell until 2 hr of incubation. After a lag, the PSV was formed in a converting cell through the process which is not a simple reversal of its degrading process.     

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.     

Prothoracicotropic Hormone Bioassay: Pupal-Adult Bombyx Assay

Blockage of adult development by brain removal and its resumption by application of the prothoracicotropic hormone (PTTH) were studied using pupae of a racial hybrid J-122 × C-115 of Bombyx mori . A log-linear dose-response relationship was obrained after injection of a PTTH solution. The Bombyx -unit of PTTH has been defined from this dose-response curve.     

ULTRASTRUCTURAL CHANGES IN EMBRYONIC ECTODERMAL CELLS OF THE NEURULATING RAT EMBRYO

Embryonic ectodermal cells of rat embryos were examined by light and electron microscopy during the early stage of neurulation. Before the onset of neurulation (day 9–6 hr embryos), the cells underwent certain characteristic ultrastructural changes; that is, apical cytoplasmic protrusions and free spherules appeared, numerous vacuoles were formed in the cytoplasm, mitochondria showed ballooning, and the endoplasmic reticulum became dilated. The amniotic cells derived from the embryonic ectoderm exhibited the same ultrastructural changes, but those from the extraembryonic mesoderm did not. Embryonic mesodermal cells and neuroectodermal cells also did not show these changes. In the middle stage of neurulation (day 9–12 hr embryos), the embryonic ectodermal cells and the amniotic cells derived from the embryonic ectoderm assumed a flat squamous shape. None of the ultrastructural changes observed in day 9–6 hr embryos were noted in these cells. The functional significance of the production of apical cytoplasmic protrusions and free spherules in the embryonic ectodermal cells and amniotic cells is discussed in relation to similar phenomena reported to occur in other cell types.     

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWING INTERACTION WITH FIXED UNFERTILIZED EGGS

The respiration of spermatozoa of the sea urchin, Hemicentrotus pulcherrimus , was found to be sensitive to rotenone, antimycin A, and cyanide. This suggests that sperm respiration results from electron transport which spans the whole mitochondrial respiratory chain. The sperm respiration was inhibited by oligomycin and this inhibition was released by 2, 4-dinitrophenol (DNP). DNP did not stimulate the respiration of spermatozoa in a diluted suspension (2 × 10⁸/ml), where they were swimming vigorously. The ADP level of spermatozoa in the diluted suspension was markedly higher than that in dry sperm. The spermatozoa, which had reacted with unfertilized eggs fixed with glutaraldehyde, were immotile with a quite low respiratory rate. The respiratory rate of the immotile spermatozoa was enhanced by DNP. In the immotile spermatozoa, ADP level was markedly low and the ATP level was as high as that in dry sperm. From these findings, it is concluded that in the swimming spermatozoa respiration coupled with oxidative phosphorylation occurs at the maximum rate. State 3 respiration probably occurs in the swimming spermatozoa. The low respiratory rate of the immotile spermatozoa is assumed to be due to a shortage of ADP and is practically regarded as state 4 respiration.     

Differentiation of Various Cell Types during Fruiting Body Formation of Dictyostelium Discoideum*

The processes of differentiation of the presumptive cells (prespore and prestalk cens) into mature spores, stalk and basal-disc cells in Dictyotelium discoideum was investigated. The number of stalk and disc cells in pre-labeled culminating cell masses was estimated by determining the radioactivity of the undissociable fraction separated by filtration from the dissociable fraction containing presumptive cells and spores. Changes in the proportion of amoeboid cells stainable with fluorescein-conjugated antispore serum and encapsulated spores were also followed in the dissociable fraction. Formation of stalk and disc cells began at 17 hr of development and was completed at 26 hr, while formation of morphologically identifiable spores began at 18 hr and was completed at 20 hr, long before completion of stalk formation. At the onset of culmination, unstained cells abruptly increased with an accompanying decrease of stained cells, when unstained rear-guard cells appeared in the hind region. Although some of the rear-guard cells soon differentiated into basal-disc cells, the rest remained amoeboid in the upper part of the spore mass (sorus) after complete formation of a fruiting body. Despite the presence of the amoeboid cells in mature sori, the proportion of the sorus to the stalk and disc of a fruiting body was approximately equal to that of stained (prespore) to unstained (prestalk) cells in a migrating slug.     

HORMONES CONTROLLING COLLAGEN SYNTHESIS DURING METAMORPHOSIS IN THE THIGH BONE OF THE TADPOLE OF RANA CATESBEIANA

The rate of ¹⁴C-proline incorporation into collagen in the thigh bone of the Rana catesbeiana tadpole was determined in vitro. Intraperitoneal injection of bovine prolactin caused an increase in the rate of collagen synthesis during the premetamorphic stages (stages 12–16) and the early metamorphic stage (stage 18), but it exerted no effect on collagen synthesis in the metamorphic stages (stages 20–25). On the other hand, injection of growth hormone stimulated the rate of collagen synthesis in the metamorphic stages and caused a slight increase in the premetamorphic stages. When a tadpole in the early premetamorphic stages (stages 12–14) was kept in 5 × 10₋₈ M thyroxine solution for several days, the rate of collagen synthesis became higher than that in the bone of the control animal. The rate of collagen synthesis was not enhanced by prolactin in the thyroxine-treated tadpole, but was stimulated by growth hormone, even when the thyroxine-treated animal remained in the premetamorphic stages. With the treatment of the tadpole by thyroxine, prolactin-sensitivity seems to be reduced, and growth hormone-sensitivity becomes apparent.     

EFFECTS OF THYROXINE AND PROLACTIN ON COLLAGEN BREAKDOWN IN THE THIGH BONE AND TAIL FIN OF THE RANA CATESBEIANA TADPOLE

Injection of ¹⁴C-proline into the tadpole causes labeling of protein in the collagen fraction of the thigh bone and tail fin. The radioactivity of the ¹⁴C-hydroxyproline residue is about 26% of the total radioactivity in the ¹⁴C-labeled protein of the collagen fraction in the thigh bone as well as in the tail fin. In ¹⁴C-proline-loaded tadpoles into which prolactin has been injected, the radioactivity in the collagen fraction in these tissues is markedly higher than that in control animals. In thyroxine-treated tadpoles, the ¹⁴C-radioactivity of the collagen fraction in the thigh bone is always higher than that of the controls, but it is markedly low in the tail fin. During the incubation of thigh bone and tail fin isolated from ¹⁴C-proline-loaded tadpoles, low molecular weight materials containing ¹⁴C-hydroxyproline are released from the ¹⁴C-labeled protein of these tissues. The rate of ¹⁴C-hydroxyproline release, which represents the rate of collagen breakdown, is higher in thigh bone and tail fin isolated from thyroxine-treated tadpoles and is markedly lower in these tissues isolated from prolactin-treated tadpoles than in those isolated from controls. In these tissues, the high rate of collagen breakdown in thyroxine-treated tadpoles is reduced by prolactin injection.     

INHIBITION OF THYROXINE-INDUCED RESORPTION OF TADPOLE TAIL BY ADENOSINE 3', 5'-CYCLIC MONOPHOSPHATE

Small segments of tail of Bufo bufo japonicus tadpoles were cultured in medium containing thyroxine (T₄) and dibutyryl cyclic AMP (dbcAMP). Like prolactin, the cyclic nucleotide blocked T₄-induced shrinkage or tail pieces. Histological study of the segments after 4-days culture revealed that dbcAMP suppressed degenerative changes induced by T₄. The inhibitory effect of prolactin on T₄-induced tail regression was promoted by caffeine, an inhibitor of adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-phosphodiesterase.
The effect of prolactin on the level of cyclic AMP in the tail was also studied in vivo . Sixty min after prolactin injection, the cyclic AMP level was 2–3 times the control value. Possible involvement of cyclic AMP in the action of prolactin, which blocks tail resorption induced by T₄, was discussed.     

Change in the Respiratory Rate of Sea Urchin Spermatozoa following Sperm-egg Interaction in the Absence of Mg2+

Spermatozoa of the sea urchin, Hemicentrotus pulcherrimus (10⁸ cells/ml), preincubated with unfertilized eggs deprived of jelly coats (more than l0⁵ cells/ml) at 20°C for 20min in Mg²⁺ free artificial sea water containing 1 mM Ca²⁺ (MFASW), exhibited very low respiration, which was enhanced by 2, 4 dinitrophenol (DNP). The fertilization rate in MFASW was usually less than 5% and was about 25% at most. Preincubation with fertilized eggs (with and without a fertilization membrane) in MFASW did not reduced the respiratory rate of spermatozoa. The rate of sperm respiration was lower in MFASW than in artificial sea water (ASW), but was higher than the respiratory rate of spermatozoa preincubated in MFASW with unfertilized eggs. Sperm respiration in MFASW or in ASW was not stimulated by 2, 4 dinitrophenol. Almost complete inhibition of sperm respiration was obtained with unfertilized eggs fixed with glutaraldehyde at concentrations of above 10⁵ cells/ml in MFASW and of about l0⁴ cells/ml in ASW. The respiratory rate of spermatozoa treated with fixed eggs was enhanced by DNP. It is concluded that the respiratory rate of the spermatozoa is reduced by their interaction with unfertilized eggs before their penetration into the eggs.