ON THE SYSTEM CONTROLLING THE TIME OF MICROMERE FORMATION IN SEA URCHIN EMBRYOS

The previously reported observation that micromere formation after cleavage suppression is not linked with the number of blastomeres present but rather with the time schedule of the fourth cleavage of the normal embryos has been confirmed. A hypothesis is advanced that a rhythmical fluctuation of the sulfhydryl contents of the egg proteins is the clock system, and micromere formation is connected with the fourth SH cycle after fertilization. The hypothesis was tested under 3 conditions:

• (i) Conditions which stop the nuclear activities but preserve the SH cycle, followed by a release from these conditions.
• (ii) Conditions which “freeze” both nuclear and cytoplasmic rhythms, and later removal of the conditions.
• (iii) Conditions which leave nuclear activities intact but prevent the cytoplasmic rhythms, followed by normal culturing.

The results came out as anticipated by the hypothesis.     

EFFECTS OF BENZIMIDAZOLE ON CLEAVAGE OF THE SEA URCHIN EGG*

Unfertilized eggs of sea urchins were treated with benzimidazole. They were fertilized after being kept in normal sea water for a certain period. It was found that the first cleavage occurred much earlier than in the control. The eggs had a tendency to cleave directly into 3 or 4 cells. Benzimidazole induced some visible changes in unfertilized eggs, which was considered to be the result of an insufficient activation. Benzimidazole was found to have the same effect as hypertonic solution has in Loeb's “double treatment” method for artificial parthenogenesis. When eggs activated with butyric acid were treated with benzimidazole instead of hypertonic solution, they cleaved in a high percentage.     

SPICULE FORMATION IN VITRO BY THE DESCENDANTS OF PRECOCIOUS MICROMERE FORMED AT THE 8-CELL STAGE OF SEA URCHIN EMBRYO*

The micromeres at the 16-cell stage of sea urchin embryo have already been endowed with a faculty to self-differentiate into spicule-forming cells (11). The present experiment was designed to test whether the factor(s) necessary for such self-differentiation had already been localized at the 8-cell stage in an area corresponding to the presumptive micromere region in Hemicentrotus pulcherrimus. Since the blastomeres at the 8-cell stage are all equal in size in normal embryo, unequal 3rd cleavage, by which small blastomeres are pinched off toward the vegetal pole (precocious micromeres), was experimentally induced either by treatment with 4NQO (4-nitroquinoline-1-oxide) at the 2-cell stage or by continuous culture in Ca-free sea water. The precocious micromeres were cultured in vitro in natural sea water containing horse serum. Descendants of the precocious micromeres formed spicules. In comparison their spicule formation with that by the descendants of the micromere of normal embryo, no differences were found regarding 1) time of initiation of spicule formation, 2) rate of growth of spicule, 3) size and shape of resultant spicule and 4) percentage of clones which formed spicule. The fact indicates that factor(s) indispensable for self-differentiation into spicule-forming cells have already been localized near the vegetal pole as early as the 8-cell stage.     

EFFECTS OF TWO PROTEASE INHIBITORS ON SEA URCHIN AND AMPHIBIAN EGG DEVELOPMENT*

After a review of our present knowledge about the effects of proteases and protease inhibitors on cell growth and egg fertilization, the results of experiments where sea urchin and amphibian eggs were treated with two protease inhibitors (TPCK, TLCK) are described. Cleavage was hardly affected, but gastrulation quickly stopped or was incomplete. The morphogenetic abnormalities which follow can be explained by abnormal gastrulation and other factors: persistence of remnants of the fertilization membrane in sea urchin larvae and dissociation of the ectoderm cells in Xenopus embryos.     

INDUCTION OF ASTER FORMATION AND CLEAVAGE IN EGGS OF THE SEA URCHIN HEMICENTROTUS PULCHERRIMUS BY INJECTION OF SPERM COMPONENTS*

Studies were made on which components of sperm were able to induce aster formation and cleavage of eggs of the sea urchin Hemicentrotus pulcherrimus. The sperm components were separated by homogenization and centrifugation into the following 3 fractions: the head-midpiece, midpiece and tail. The head-midpiece fraction was then divided into 2 sub-fractions, the centriole sub-fraction and the centriole-free sub-fractions. Each fraction was injected into unfertilized eggs and after 15–30 min the eggs were inseminated. The ability of a fraction or a sub-fraction to induce aster formation and cleavage was deduced from the frequency of multipolar cleavage. The head-midpiece fraction and the centriole sub-fraction were effective in inducing aster formation and cleavage, but the other fractions were not. It was concluded that isolated centrioles from sea urchin sperm act as division centers in the egg.     

MOLECULAR PROPERTIES AND DIFFERENTIATION OF ACETYLCHOLINESTERASE IN SEA URCHIN EMBRYOS*

The two molecular forms of acethylcholinesterase (EC 3.1.1.7) in sea urchin embryos were characterized by several physical methods. The sedimentation coefficients determined by sucrose gradient centrifugation are 7.6S and 10.6S. The Stokes radii determined by gel filtration are 65 Å and 91 Å. From these parameters, molecular weights were estimated as 190,000 and 380,000; the one is twice as large as the other. Both forms have similar electric property and buoyant density in a CsCl gradient. When the enzyme solution was concentrated, the 10.6S form became predominant. These results suggest that the two forms are monomer and dimer. The sea urchin enzymes resemble globular forms of acetylcholinesterase of the electric organ of fishes. The activity of the enzyme abruptly increases in post-gastrulation embryos. Inhibition of concomitant protein synthesis by a specific inhibitor, emetine, does not affect the increase in enzyme activity. The result suggests that post-translational processes may be involved in the differentiation of this enzyme in sea urchin development. The following sea urchins were used in the study: Strongylocentrotus purpuratus, Strongylocentrotus franciscanus, and Dendraster excentricus.     

LOCALIZATION OF DYNEIN IN SEA URCHIN EGGS DURING CLEAVAGE*

Detection and localization of dynein in cleaving sea urchin eggs were attempted using antidynein serum (prepared against a tryptic fragment of dynein, Fragment A, of sea urchin sperm flagella) and fluorescein conjugated goat antiserum to rabbit γ-globulin. In both unfertilized and newly fertilized eggs, fluorescence was distributed rather uniformly within the cells but was absent from the nuclei. At prophase, intense fluorescence was observed on both sides of nucleus, suggesting accumulation of dynein in developing asters. From metaphase to anaphase, the whole mitotic apparatus (MA) was stained with the exceptions of the chromosomes and pole areas. Fluorescence then again became dispersed within the eggs. Throughout the mitotic process and cytokinesis, the egg cortex including the cleavage furrow was stained intensely, presumably reflecting the presence of dynein in this region. Similar distributions of fluorescence were obtained with the isolated MAs. Neither non-immune serum nor the antiserum to which Fragment A was absorbed stained the eggs. Little staining was obtained with the antiserum against starfish egg myosin. The results, together with the finding that the chromosome motion in the isolated MAs was completely inhibited by anti-dynein serum, but not with the anti-myosin serum, suggest an active role played by a tubulin-dynein system in mitosis.     

FORMATION OF THE CORTICAL CONCAVITY AT FERTILIZATION IN THE SEA URCHIN EGG

During the initial stages of fertilization envelope elevation in eggs of Strongylocentrotus pur puratus and S. droebachiensis a large concavity of the egg cortex was observed in the light microscope. This concavity corresponded in shape and size with the elevating fertilization envelope. However, after the vitelline layers of eggs were disrupted and the eggs inseminated, the concavity failed to develop although the eggs were fertilized and developed normally. We propose that the concavity is formed owing to increased hydrostatic pressure within the perivitelline space. To further support this hypothesis we measured total egg protein secreted during fertilization, and found that 98% was retained within the perivitelline space. Furthermore, 80% of the total protein was contributed by the hyaline layer. Presumably, colloidal osmotic pressure and/or hydration of fertilization product, trapped beneath the fertilization envelope, is responsible for increased hydrostatic pressure within the perivitelline space, and therefore promotes not only fertilization envelope elevation, but the cortical concavity as well.     

ENZYMES OF THE TRICARBOXYLIC ACID CYCLE IN SEA URCHIN EGGS AND EMBRYOS

Changes in the activity of some enzymes of the tricarboxylic acid cycle during development of sea urchins were investigated. Unfertilized eggs showed substantial activity of citrate synthase, aconitase, NAD- and NADP-specific isocitrate dehydrogenases, fumarase and malate dehydrogenase. During development, the activity of citrate synthase, aconitase, NADP-specific isocitrate dehydrogenase and malate dehydrogenase increases gradually, whereas the activity of fumarase remains rather constant. There is no close correlation between changes in the enzyme activity and the increase in oxygen consumption during development. Citrate synthase, aconitase, NADP-specific isocitrate dehydrogenase are mainly localized in the mitochondrial fraction, whereas fumarase and malate dehydrogenase are present in both mitochondrial and cytosol fractions. The intracellular localization of these enzymes does not change during development. A possible mechanism for the regulation of some enzymes of the tricarboxylic acid cycle in sea urchin eggs is discussed.     

FORCE EXERTED BY THE CLEAVAGE FURROW OF SEA URCHIN EGGS

A drop of ferrofluid injected into the center of a dividing sea urchin egg is deformed into the shape of an hourglass when the cleavage furrow advances. The force applied to the drop is determined from the deformation of the drop and the interfacial tension between the ferrofluid and the protoplasm. The interfacial tension is determined from the deformation of a spherical drop in the protoplasm when a magnetic field is applied, and the force applied to the drop, which is estimated from the deformation by magnetic field of a similar drop in 2 per cent aqueous solution of Triton X-100 and the interfacial tension between the ferrofluid and this solution.
The force applied to the drop in the dividing egg increases during an early stage of cleavage and decreases during a later stage. The force attained a maximum of 9 × 10⁻³ dyne in an egg of Temnopleurus toreumaticus which pinched the drop into two when it divided. Smaller maximum forces, 3.9 × 10⁻³ dyne in the eggs of Temno-pleurus toreumaticus and 2.0 × 10⁻³ dyne in the eggs of Clypeaster japonicus (mean values), were obtained when the furrowing was arrested by the drop. The magnitude of the maximum tension developed in the contractile element located in the furrow cortex is discussed.     

INHIBITION OF DIHYDROFOLATE REDUCTASE BY PALMITOYL-CoA AND THE REVERSAL OF THE INHIBITION BY SPERMINE AND SPERMIDINE IN THE EGGS OF THE SEA URCHIN,HEMICENTROTUS PULCHERRIMUS*

Dihydrofolate reductase activity in fertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, was almost the same as in unfertilized eggs. Aminopterin inhibited the enzyme competitively with dihydrofolate (FH₂). The apparent K_m value for FH₂ in the dihydrofolate reductase reaction was about 0.1 μM in the crude homogenate of both unfertilized and fertilized eggs. Dihydrofolate reductase in the eggs was also inhibited by palmitoyl-CoA. The inhibition was canceled by polyamines, especially by spermine, but putrescine failed to prevent the enzyme from the inhibition. The change in long-chain acyl-CoA and polyamine concentrations during fertilization are discussed as possible regulatory factors of the enzyme.     

EFFECTS OF CHEMICAL REAGENTS ON THE CYCLIC CHANGES OF CORTEX AND CYTOPLASM IN THE ACTIVATED ENUCLEATED EGG-FRAGMENTS OF THE SEA URCHIN*

Unfertilized eggs of sea urchins. Anthocidaris crassispina and Hemicentrotus pulcherrimus, were separated by centrifugation into two fractions (nucleated light and enucleated heavy fragments). The enucleated egg-fragments were activated by treatment with 1 M urea and then put into sea water solutions of the following three reagents; colcemid, cytochalasin B and Monogen at a concentration by which cleavage was suppressed. It was then examined whether the egg-fragments can exhibit cyclic changes of cytoplasm and cortex in correlation with the cleavage cycle in normally fertilized eggs without any influence of nuclear activity. The results obtained clearly showed that colcemid can suppress the cyclic appearance of cytoplasmic changes, but not that of cortical changes; on the contrary, in cytochalasin B- and Monogen-treated fragments, the periodicity in cortical activities is suppressed, while the periodic changes in the cytoplasm appear according to a timeschedule of the cleavage cycle. Therefore, it may be said that: 1) cyclic changes can occur in both the cytoplasm and the cortex independently, without the direct influence of nuclear activity; 2) if either of them is arrested, the cleavage does not take place; 3) the normal cleavage requires the simultaneous occurrence of periodic activities both in the cortex and in the cytoplasm after fertilization.     

SOME STRUCTURAL AND FUNCTIONAL ASPECTS OF THE MITOTIC APPARATUS IN SEA URCHIN EMBRYOS

The mitotic figures in dividing cells of sea urchin embryos, from first division to the onset of cilia formation, were studied with regard to the filament system and its relation to kinetochores, chromosomes, and poles, as well as to fixation conditions which would best preserve these structures. With regard to fixation, variations in the salt concentration and pH of the fixative indicated that an extraction effect on the chromosomes noted in earlier work was probably due to a combination of neutral pH and salt concentration equivalent to sea water. The presence of the 15 mµ filaments depended on the presence of either of two stabilizing conditions: pH 6.1 or presence of the salts of sea water, presumably the divalent cations of Ca and Mg. Kinetochores and centrioles were unaffected by the fixative variations. The 15 mµ filaments, reported earlier in the central spindle, are also found in great numbers in the asters of early cleavage divisions. However, with successive divisions and reduction in cell size, the aster disappears at about the 32 to 64 cell stage, and the 15 mµ filaments are entirely associated with the central spindle. This disappearance of the aster suggests that it may be, in fact, merely a specialization of large cells for cytokinesis.     

DEPENDENCE OF PERIODIC DNA SYNTHESIS ON PROTEIN SYNTHESIS: DELAY OF DNA SYNTHESIS IN THE EARLY CLEAVAGE STAGE OF SEA URCHIN EMBRYOS TREATED WITH CYCLOHEXIMIDE AND COLCHICINE

The effects of cycloheximide and colchicine on cleavage and syntheses of DNA and proteins in cleaving embryos of the sea urchin, Hemicentrotus pulcherrimus , were examined. Cycloheximide caused delay of cell division with prolongation of the streak stage. Both inhibitors also caused delay in initiation of DNA synthesis. The decrease in the rate and prolongation of the period of DNA synthesis caused by these inhibitors varied with their concentrations and the time of administration. Initiation of DNA synthesis was delayed when cycloheximide was added to suspensions of embryos between the time after preceding DNA synthesis terminated and a definite time before the predicted time of initiation of the next synthesis of DNA, except at the stage of pronuclear fusion. However, when the inhibitor was added after initiation of the synthesis, the latter proceeded normally. Addition of 10 m m cycloheximide immediately after fertilization or 2 m m cycloheximide 60 min before fertilization also delayed DNA synthesis at the stage of pronuclear fusion, indicating that synthesis at this stage also required prior protein synthesis. Colchicine had less inhibitory effect on protein synthesis, but greatly delayed initiation of DNA synthesis and prolonged its duration. These facts suggest that a definite amount of a particular protein must be synthesized and accumulated in each synthetic cycle before initiation of DNA synthesis.     

LOCALIZATION AND MULTIPLE FORMS OF ACETYLCHOLINESTERASE IN SEA URCHIN EMBRYOS

Acetylcholinesterase during the development of the sea urchin Pseudocentrotus depressus was examined by enzyme assay (the colorimetric method of E llman et al. ), histochemistry (a Cu-thiocholine method), polyacrylamide gel electrophoresis and DEAE-Sephadex ion exchange chromatography.
The enzyme activity is detected in the unfertilized egg, remains low during cleavage, elevates slightly through gastrulation, and then increases rapidly thereafter. The intense activity is localized in the mesenchyme cells associated with the larval skeleton of young pluteus larvae, and their cell membranes and nuclear envelops. Soluble enzyme accounts for 60% of the total activity. The additional 34% is extracted by 1% Triton X-100 from particulates. The soluble enzyme consists of two forms. Both are strongly acidic proteins which are similar in electric charge, but dissimilar in size, being 180,000 and 280,000 in molecular weights. The enzyme released from the membrane by the detergent possesses a component which is not present in the soluble complement of the enzyme. It is not a secondary product of the soluble enzyme interacting with the detergent.
Acetylcholinesterase serves as a marker of late differentiation and regional differentiation in the sea urchin embryo.     

ON THE DE NOVO FORMATION OF THE CENTRIOLE IN THE ACTIVATED SEA URCHIN EGG

Eggs of Pseudocentrotus depressus were activated artificially by Loeb's double treatment method. 50 min after activation, a number of asters were produced in the eggs. It was confirmed by electron microscopy that centrioles with a typical fine structure were present in artificially induced asters.
An unfertilized egg of Hemicentrotus pulcherrimus was divided into 2 halves, nucleated and non-nucleated, by centrifugation on a sucrose bed. Each half was activated by the same method as mentioned above. Several asters were produced in both halves after a certain period of incubation. The presence of bodies considered to be centrioles were demonstrated in the asters in both nucleated and non-nucleated halves.
The results add probability to the view that the centrioles are produced de novo in artificially activated eggs and fragments.     

FORMATION AND BEHAVIOR OF PINOSOMES IN THE SEA URCHIN OOCYTE DURING OOGENESIS

Formation and behavior of the pinosomes at the surface of the oocyte during oogenesis in the 4 species of sea urchins, Anthocidaris crassispina, Temnopleurus toreumaticus, Mespilia globulus and Pseudocentrotus depressus, were studied. The plasma membrane of the oocyte is almost smooth at the early stage of oogenesis, although a small number of cytoplasmic processes appear on it, facing the germinal epithelium. At the beginning of vitellogenetic stage many processes appear on the whole surface of the oocyte. Near the base of the fully grown process, the pinosome designated as the α-pinosome is formed. The α-pinosome may play a part in maturation of the yolk granule. The processes shorten as a whole at the time of the breakdown of the germinal vesicle. Formation of the pinosome designated as the β-pinosome begins just before vitellogenetic stage and continues during this stage. The β-pinosome may be directly concerned with the formation of cortical granules.     

STABILITIES OF NUCLEAR AND MESSENGER RNA MOLECULES IN SEA URCHIN EMBRYOS

The kinetics of accumulation of radioactive adenosine in adenosine triphosphate and in RNA of nuclear, cytoplasmic, and polysomal fractions of sea urchin embryos have been analyzed. 85% of the RNA synthesized decays in the nucleus with an apparently uniform half-life of about 7 min. The remaining 15% goes to the cytoplasm, mostly entering polysomes, and decays with a quite uniform half-life of about 75 min. The nuclear RNA accounts for one-third and the cytoplasmic RNA accounts for two-thirds of the total unstable RNA which accumulates at steady state in the embryo. The size distribution of short-labeled nuclear RNA is very similar to that of long-labeled messenger RNA, when both are extracted directly from the cells without a previous cell fractionation.     

THE EFFECTS OF NICOTINE ON FERTILIZATION IN THE SEA URCHIN, ARBACIA PUNCTULATA

The number of sperm incorporated into eggs made polyspermic with varying concentrations of nicotine (0.025–0.25%, v/v) appears to be directly related to the concentrations employed. The cortical response is morphologically equivalent to that observed in control preparations. Shortly after their incorporation all of the spermatozoa undergo structural events normally associated with the development of the male pronucleus in monospermic eggs. During the reorganization of the spermatozoa, sperm asters are formed. The number of male pronuclei that initially migrate to and encounter the female pronucleus is usually one to three. When pronuclei come into proximity to one another the surface of the female pronucleus proximal to the advancing male pronuclei flattens and becomes highly convoluted. Subsequently, the pronuclei contact each other and the outer and inner membranes of the pronuclear envelopes fuse, thereby producing the zygote nucleus. The male pronuclei remaining in the zygote after this initial series of pronuclear fusions continue to differentiate, i.e. they enlarge, form nucleolus-like bodies, and undergo further chromatin dispersion. In approximately 90% of the zygotes, all of the remaining male pronuclei progressively migrate to the zygote nucleus and fuse to form one large nucleus by 80 min postinsemination. Mitosis and cleavage of the polyspermic zygote occurs later than in monospermic eggs.