INITIATION OF CLEAVAGE IN FISH EGGS BY INJECTION OF FLAGELLA OR MICROTUBULES OF SEA URCHIN SPERMATOZOA

Flagella and their microtubules obtained from sea urchin ( Hemicentrotus pulcherrimus ) spermatozoa were injected into unfertilized eggs of the medaka ( Oryzias latipes ) with a micropipette. Upon activation, some of the eggs began the first cleavage with three or more irregular blastomeres, and developed to the morula stage. It is suggested that sperm flagellar microstubule material is one of the cleavage initiation substances.     

False fertilization in sea urchin eggs induced by diabolin, a 120K kelp protein

A 120K lectin-like protein was isolated from the kelp Laminaria diabolica (Oni-kombu), with a unique activity to induce false fertilization specifically in the eggs of the sea urchin Hemicentrotus pulcherrimus. The protein designated as "diabolin" rendered the unfertilized egg forms and elevated the fertilization envelope without insemination at 18 nM half-maximally. Those eggs with elevated fertilization envelopes, however, could not enter into normal cleavage or further development, and hence the proliferation of the sea urchin was hindered. Diabolin, thus, by its unique defense mechanism protects the kelp from the predator sea urchin. It was partially sequenced and found to have the highest homology with phytoene dehydrogenase from the plant virus Erwinia uredovora. A question was left to be solved as to how the kelp on the southeast coast of Hokkaido Island could develop the defense mechanism against the sea urchin on Honshu Island separated by Tsugaru Straits.     

Cytological effects of urethan on cleavage induction in parthenogenetically activated sea urchin eggs

The effect of urethan on artificial induction of cleavage in eggs of the sea urchin, Hemicentrotus pulcherrimus, was studied. When the eggs were exposed for 20 minutes to seawater containing urethan (final concentration, 0.08 M) after butyric acid-activation and then treated with hypertonic seawater, the cleavage rate was enchanced by about 1.5 times as compared with the nontreated eggs. In the eggs exposed to urethan–seawater for over 70 minutes many clear spots appeared throughout the cytoplasm. Simultaneously, the pigment granules, which had been embedded within the cortex, migrated to the inner cytoplasm and encircled a monastral centrosphere and clear spots. The clear spots were composed of microtubules much like cytasters, and in the central region of them centrioles were not yet found. The eggs in which the pigment granules disappeared from the cortex may be more susceptible to cleavage induction by succeeding hypertonic treatment.     

Modulation of calcium sensitivity by a specific cortical protein during sea urchin egg cortical vesicle exocytosis

A study of the Ca2+ sensitivity of cortical vesicle (CV) discharge has been accomplished using isolated sea urchin egg cortices. Cortices isolated in a medium ionically similar to normal egg cytoplasm discharge 50% of their CVs at 1.6 microM Ca2+ (=[Ca2+]50). Alternatively, cortices isolated in a medium containing 500 mM chaotropic anions (Cl-, Br-, I-, or NO-3) discharge their CVs at 16 microM [Ca2+]50. Incubation with the 500 mM KCl extract of cortices restores high Ca2+ sensitivity and the mode of CV discharge characteristic of cortices before extraction. Fractionation of egg homogenates by differential centrifugation reveals that about 20% of the total restoring activity is associated with the cortex. In eggs of Hemicentrotus pulcherrimus, the factor responsible for this restorative function is a heat and protease labile protein with a molecular weight of 100,000. Similar activity is seen also in the eggs and sperm of other species of sea urchin.     

The isotopic effects of D2O in developing sea urchin eggs

When developing sea urchin eggs were treated with sea water containing 40% D2O (D2O-SW) at the 8-cell stage, the micromere formation was delayed and micromeres were larger than those seen in the control. But eggs returned to normal artificial sea water (NASW) at the 16- to 32-cell stage did not form abnormal spicules in larvae of Pseudocentrotus depressus. Little effect on the spicule formation of Hemicentrotus pulcherrimus was also noted. If the culture period in D2O-SW was extended until the hatching stage, the number of plutei with abnormal spicules increased. Primary mesenchyme cells of Pseudocentrotus depressus larvae failed to make two aggregated spicule rudiments on the ventral side of the larva and developed a ring-like spicule. This ring-like spicule, however, only occasionally occurred in the larvae of Hemicentrotus pulcherrimus. The cell cycle was longer in the presence of D2O. However, blastomeres managed to divide throughout the development. Larvae reared in 20% D2O-SW after the hatching stage developed into quasi-normal plutei but smaller than control. We found no exogastrulation in these larvae. Exogastrulation was found only in larvae continuously cultured in 40% D2O-SW from the early development. These results are inconsistent with previous reports made by other authors.     

The amino acid sequence, immunofluorescence and microinjection studies on the 15 kDa calcium-binding protein from sea urchin egg

The 15 kDa protein is the most abundant low molecular weight Ca2+-binding protein, different from calmodulin, in eggs of sea urchin, Hemicentrotus pulcherrimus. The data from the amino acid sequence demonstrated that the 15 kDa protein belonged to the troponin C superfamily. Based on immunofluorescent and immunomicroscopic observations, we showed that the 15 kDa protein localized in the nuclei of fertilized eggs and mitotic apparatus of dividing eggs. Microinjection of the antibody against 15 kDa protein into sea urchin blastomeres resulted in the arresting of cell division. These results suggest that the 15 kDa protein plays an important role in mitosis of sea urchin egg.     

Purification of Acetylcholinesterase from Sea Urchin (Hemicentrotus pulcherrimus) Embryos by Affinity Chromatography

Only one form of acetylcholinesterase (AchE) was detected in Hemicentrotus pulcherrimus embryos. In H. pulcherrimus embryos as well as in the other sea urchin embryos, AchE activity begins to increase rapidly after gastrula stage.
Purification of AchE from plutei has been carried out by the procedure including affinity chromatography. Purified AchE had the activity 14,600 times higher than that of homogenate, and the final yield of AchE was 8%. The enzyme seems to be electrophoretically homogeneous, and has a molecular weight of 3 × 10⁵ as determined by Sepharose CL–6B column chromatography.     

GLYCOGEN METABOLISM AND CHANGES IN THE ACTIVITIES OF PHOSPHORYLASE, PHOSPHOFRUCTOKINASE AND PYRUVATE KINASE DURING DEVELOPMENT OF SEA URCHIN EGGS

The glucose and glycogen contents of sea urchin eggs and embryos were measured enzymatically. Unfertilized eggs of Hemicentrotus pulcherrimus and Anthocidaris crassispina contain about 20.9 and 24.4 μg of glycogen per mg protein, respectively. As for glucose, unfertilized eggs of Hemicentrotus and Anthocidaris contain about 0.7 and 1.9 μg per mg protein, respectively. Glycogen consumption during embryonic development differs with different species of sea urchins. In Anthocidaris , glycogen decreases significantly after fertilization. The oxidation of glucose and glycogen accounts for about 50% of oxygen consumed until the early blastula stage in this species. The contribution ratio of glucose and glycogen to the overall energy pool becomes less than 10% at later stages. In Hemicentrotus , however, the glycogen content remains unchanged until the early blastula stage and thereafter decreases. The importance of glucose and glycogen as an energy fuel seems little throughout the development of Hemicentrotus. Activities of phosphorylase (EC 2.4.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) were measured at various embryonic stages in both species of sea urchins. The difference between two species in the consumption of glucose and glycogen can not be elucidated by the differences in the activities of these enzymes.     

Changes of tropomyosin isoforms during development of cross-fertilized sea urchin embryos

Changes of tropomyosin isoforms during development of the sea urchin, Hemicentrotus pulcherrimus , were investigated using two-dimensional urea-shift gel electrophoresis. Tropomyosin isoforms included in the embryos were gradually increased after 2 cell stage and retained at a constant level after gastrula stage. To detect the tropomyosin isoforms derived from zygotic genomes, embryos cross-fertilized between H. pulcherrimus and Pseudocentrotus depressus gametes were prepared. Since tropomyosin isoforms from H. pulcherrimus eggs and from P. depressus eggs could be distinguished from each other on a two-dimensional electrophoretic gel, the paternal isoforms of tropomyosin in the cross-fertilized embryos, which were not included endogenously in the egg, could be regarded as products derived from zygotic genomes. The paternal isoforms of tropomyosin were detected first at around the gastrula stage in embryos cross-fertilized between H. pulcherrimus sperm and P. depressus eggs and also in the reverse combination of the gamete species. Muscle tropomyosins derived from H. pulcherrimus and P. depressus genomes were similarly detected in cross-fertilized embryos at the pluteic stage when the muscle tropomyosin appeared in sea urchin embryos.     

Induction of Fertilization Membrane Formation and Cyanide-insensitive Respiration in Sea Urchin Eggs by the Treatment with Dimethylsulfoxide Followed by an Incubation in an Ice Bath

In unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus , fertilization membrane formation was induced by an incubation with dimethylsulfoxide (DMSO) for several min at 20°c followed by another incubation in an ice bath. The number of eggs with fertilization membrane, thus obtained, increased in relation to the concentration of DMSO between 1 and 3% (v/v) and was higher than 75% at concentrations above 3%. Fertilization membrane formation by this treatment occurred in Ca²⁺ free- or Ca²⁺, Mg²⁺ free- artificial sea water containing EGTA (50 mM) and was inhibited by verapamil. In the presence of DMSO, the membrane formation was also induced by 2, 4-dinitrophenol or cyanide in considerable number of eggs at 20°c. Eggs remained fertilizable, even when they were kept with DMSO for 1 hr at 20°c. DMSO slightly enhanced respiratory rate in unfertilized eggs and substantially reduced it in fertilized eggs. DMSO-treated eggs exhibited cyanide-insensitive respiratory burst following chilling in an ice bath or by adding DNP or cyanide, in a similar manner to the burst induced by sperm.     

Multiple molecular forms of acid nitrophenyl phosphatase in sea urchin eggs and embryos

Acid nitrophenyl phosphatases from sea urchin eggs and embryos were investigated by gel filtration. Four different forms were found in Hemicentrotus pulcherrimus, and three forms in Anthocidaris crassispina and Pseudocentrotus depressus. The first and second forms (designated AcP-1 and AcP-2) had the highest activity in the range of pH 5.6–6.0. The third (designated AcP-3) had an apparent optimum pH between pH 4.3 and 4.8, and the fourth (designated AcP-4) showed the maximum activity at pH 3.0. AcP-1 was much more thermolabile than AcP-2 and AcP-3 at 56°C. NaF inhibited AcP-2, AcP-3, and AcP-4 but not AcP-1. AcP-1, AcP-2, and AcP-3 were observed in the three species, whereas AcP-4 was not detected in A. crassispina and P. depressus. AcP-1, AcP-2, and AcP-3 were separted by gel filtration. AcP-1 and AcP-2 of A. crassispina and H. pulcherrimus were studied in developing embryos. The behavior of these forms in gel filtration changed during development, from unfertilized eggs to the pluteus stage.     

马粪海胆对环境变化的耐受性与选择性研究

对采自青岛近岸的马粪海胆进行温度、盐度、光强与底质等因子的耐受及选择实验。结果表明,青岛近岸马粪海胆的适温范围约在8-22℃,对温度的选择受驯养水温的影响;属窄盐性种类,适盐范围约在30-35;喜好弱光环境,饥饿状态的选择光强（8-25lx）较非饥饿状态（10-35lx）低;对粗沙砾底质具明显的正选择,而对细沙性底质呈明显的负选择。     

Distributional Change of Membrane-associated Calcium in Sea Urchin Eggs during Fertilization

The "source and sink" for the intracellular calcium released during fertilization were examined in sea urchin eggs, Hemicentrotus pulcherrimus , with chlortetracycline as a fluorescent chelate probe. In order to distinguish the differential distribution of membrane-associated calcium in various compartments in cytoplasm, eggs were stratified by centrifugation before or after fertilization. Only the layer containing mainly mitochondria exhibited the chlortetracycline-fluorescence in unfertilized eggs. After fertilization, a new fluorescent band emerged in the membrane-rich clear layer of stratified eggs. Chlortetracycline-fluorescence in the clear layer was gradually redistributed surrounding the prophase nucleus and then incorporated into the mitotic apparatus. From these observations, we postulate that the major source(s) of released free calcium ions at fertilization is in the mitochondira layer and membranes in the clear layer are newly activated as the calcium sequestering system after fertilization.     

Degradation of yolk proteins in sea urchin eggs and embryos

Yolk granules isolated from unfertilized and fertilized eggs of the sea urchins, Hemicentrotus pulcherrimus and Anthocidaris crassispina, were incubated in acidic media, and the protein components were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By the incubation, a protein (molecular weight 180,000 in H. pulcherrimus and 178,000 in A. crassispina) most abundant in unfertilized eggs decreased, while proteins (molecular weight 61,000, 72,000, 94,000, 114,000 in H. pulcherrimus and 56,000, 70,000, 92,000, 112,000 in A. crassispina) dominant in developed embryos increased. Neither alkaline nor neutral condition resulted in such changes in the electrophoretic patterns of proteins as observed in acidic media. Experiments with various inhibitors of proteases suggested that thiol protease(s), such as cathepsin B, may be the most important enzyme(s) in the degradation of yolk proteins in embryogenesis of the sea urchin.     

Immunocytochemical study of the distribution of a ganglioside in sea urchin eggs.

An antibody against M5 ganglioside (NeuGc alpha 2-6Glc beta 1-1Cer), the dominant ganglioside in the eggs of the sea urchin, Anthocidaris crassispina, was purified by affinity chromatography from rabbit antiserum against crude ganglioside of the eggs. The specificity of the antibody was verified by enzyme-linked immunosorbent assay and TLC immunostaining. M5 ganglioside was also the major one in the eggs of another sea urchin, Hemicentrotus pulcherrimus, as judged from TLC analyses including immunostaining. Cryostat-sections of H. pulcherrimus eggs were examined to determine the distribution of M5 ganglioside by indirect immunofluorescence microscopy with the antibody. Before fertilization, the egg cortex was highly stained, while the other part of cytoplasm was uniformly but much more weakly stained. After fertilization, the staining rapidly decreased in the cortex and was restricted to a very thin peripheral layer and to cytoplasmic patches. The immunoreactivity was also observed in the esophagus and the somatic cells of the testis, but the spermatozoa were never stained with the antibody.     

Mitotic Asters Separate, although Chromosomes Do Not Separate at Slightly Acidic pHi in the Fertilized Egg of the Sea Urchin, Hemicentrotus pulcherrimus

The effect of slightly acidic intracellular pH (pHi) on the development of the sea urchin, Hemicentrotus pulcherrimus was investigated. At first cleavage, the fertilized eggs were treated with artificial sea water containing sodium acetate (Ac-pHSW) at pH 6.8 or 7.0 at the onset of nuclear envelope breakdown, and their pHi decreased from 7.30 to 6.68 or 6.78, respectively. When the eggs were observed after fixation by indirect immunofluorescence and differential interference contrast microscopy, the mitotic stage of the treated eggs was arrested at metaphase and the mitotic apparatus was maintained until more than 50 min after the treatment, although it was smaller in size than that of non-treated eggs. On the other hand, the number of the mitotic asters increased from 2 to 3-4, and further to 6-8 following prolonged exposure, suggesting that the centrosomes had divided and replicated. These results suggest that the centrosome cycle advanced at slightly acidic pHi, even when the mitotic cycle did not advance beyond metaphase.     

Substructure of sea urchin egg cytoplasmic dynein

The substructure of the cytoplasmic dynein molecule was studied using the quick-freeze, deep-etch technique. Cytoplasmic dynein purified as a 12 S form from the eggs of the sea urchin Hemicentrotus pulcherrimus was composed of a single high molecular weight polypeptide. Rotary shadowing images of cytoplasmic dynein either sprayed on to a mica surface or quick-frozen on mica flakes demonstrated a single-headed molecule, in contrast to the two-headed molecule of sea urchin sperm flagellar 21 S dynein. More detailed substructure was visualized by rotary shadowing after quick-freeze deep-etching. Cytoplasmic dynein consisted of a head and a stem. The head was pear-shaped (16 nm X 11 nm) and a little smaller than the pear-shaped head of 21 S dynein (18 nm X 14 nm). The form of the stem was irregular, and its apparent length varied from 0 to 32 nm. Binding of cytoplasmic dynein to brain microtubule in the solution was observed by negative staining, and that in the precipitate was examined by the quick-freeze, deep-etch method as well. Both methods revealed the presence of two kinds of microtubules, one a fully decorated microtubule and the other a non-decorated microtubule. Cytoplasmic dynein bound to microtubule also appeared as a globular particle. Neither the periodic binding nor the crossbridges that were observed with 21 S dynein were formed by cytoplasmic dynein, although cytoplasmic dynein appeared to bind to microtubules co-operatively.     

Purification, characterization, and assembly properties of tubulin from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus

Tubulin was purified from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus by chromatography of an egg supernatant fraction on DEAE-Sephacel or DEAE-cellulose followed by cycles of temperature-dependent microtubule assembly and disassembly in vitro. After two assembly cycles, the microtubule protein consisted of the alpha- and beta-tubulins (greater than 98% of the protein) and trace quantities of seven proteins with molecular weights less than 55 000; no associated proteins with molecular weights greater than tubulin were observed. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on urea-polyacrylamide gradient gels, the alpha- and beta-tubulins did not precisely comigrate with their counterparts from bovine brain. Two-dimensional electrophoresis revealed that urchin egg tubulin contained two major alpha-tubulins and a single major beta species. No oligomeric structures were observed in tubulin preparations maintained at 0 degrees C. Purified egg tubulin assembled efficiently into microtubules when warmed to 37 degrees C in a glycerol-free polymerization buffer containing guanosine 5'-triphosphate. The critical concentration for assembly of once- or twice-cycled egg tubulin was 0.12-0.15 mg/mL. Morphologically normal microtubules were observed by electron microscopy, and these microtubules were depolymerized by exposure to low temperature or to podophyllotoxin. Chromatography of a twice-cycled egg tubulin preparation on phosphocellulose did not alter its protein composition and did not affect its subsequent assembly into microtubules. At concentrations above 0.5-0.6 mg/mL, a concentration-dependent "overshoot" in turbidity was observed during the assembly reaction. These results suggest that egg tubulin assembles into microtubules in the absence of the ring-shaped oligomers and microtubule-associated proteins that characterize microtubule protein from vertebrate brain.     

Purification of a Sperm Lectin Extracted from Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus

Hemagglutinating activity for human type A erythrocytes was detected in a sperm extract obtained by treatment with Triton X-100 of spermatozoa from the sea urchin Hemicentrotus pulcherrimus. Among tested sugars only N-acetyl-D-galactosamine had any inhibitory effect on the hemagglutinating activity of the sperm extract. The lectin was purified by a combination of affinity chromatography and ion-exchange chromatography. A single band was obtained after SDS-polyacrylamide gel electrophoresis of the purified lectin, corresponding to an apparent molecular weight of 15,000 daltons. Trypsin-generated fragments of the surface of eggs significantly inhibited hemagglutination of erythrocytes by the purified lectin. The biological role of the sperm lectin is discussed.