Pseudocentrotus Depressus Research Articles

Reaction mechanism of 21S dynein ATPase from sea urchin sperm. I. Kinetic properties in the steady state.

21S Dynein ATPase [EC 3.6.1.3] from axonemes of a Japanese sea urchin, Pseudocentrotus depressus, and its subunit fractions were studied to determine their kinetic properties in the steady state, using [gamma-32P]ATP at various concentrations, 5 mM divalent cations, and 20 mM imidazole at pH 7.0 and 0 degrees C. The following results were obtained. 1. 21S Dynein had a latent ATPase activity of about 0.63 mumol Pi/(mg . min) in 1 mM ATP, 100 mM KCl, 4 mM MgSO4, 0.5 mM EDTA, and 30 mM Tris-HCl at pH 8.0 and 25 degrees C. Its exposure to 0.1% Triton X-100 for 5 min at 25 degrees C induced an increase in the ATPase activity to about 3.75 mumol Pi/(mg . min) and treatment at 40 degrees C for 5 min also induced a similar activation. 2. The double-reciprocal plot for the ATPase activity of dynein activated by the treatment at 40 degrees C consisted of two straight lines, while that of nonactivated 21S dynein fitted a single straight line. 3. In low ionic strength solution, the Mg- and Mn-ATPase of 21S dynein showed substrate inhibition at ATP concentrations above 0.1 mM; the inhibition decreased with increasing ionic strength. Ca- and Sr-ATPase showed no substrate inhibition. 4. Both the Vmax and Km values of dynein ATPase decreased reversibly upon addition of about 40% (v/v) glycerol. In the presence of glycerol, the dynein ATPase showed an initial burst of Pi liberation. The apparent Pi-burst size was 1.0 mol/(10(6) g protein) and the true size was calculated to be 1.6 mol/1,250 K after correcting for the effect of Pi liberation in the steady state and the purity of our preparation. 5. One of the subunit fractions of 21S dynein which was obtained by the method of Tang et al. showed substrate inhibition and an initial burst of Pi liberation of 1.4 mol/(10(6) g protein) in the presence of 54% (v/v) glycerol.

Ribosomal proteins of sea urchin eggs

Ribosomal proteins from unfertilized eggs of three sea urchin species. Pseudocentrotus depressus, Hemicentrotus pulcherrimus, and Anthocidaris crassispina, were analyzed. Species-specific differences were observed in the profiles of large subunit proteins on two-dimensional slab gels, though the number of ribosomal proteins and the molecular weights of their counterparts were the same. The small subunit proteins revealed similarities in the electrophoretic profiles and in the phosphorylation patterns among these three species.

Ribosomal proteins of sea urchin eggs. I. Characterization of cytoplasmic ribosomal proteins

Ribosomal proteins from the eggs of sea urchin, Pseudocentrotus depressus, were analyzed by two-dimensional polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. Thirty different proteins including three acidic proteins were detected in the small subunit. The large subunit was found to contain at least 43 different proteins, 42 basic and one acidic. The ribosomal proteins from the small subunit have molecular weights ranging from 11,900 to 51,700 (average molecular weight: 25,600). The molecular weights of the large subunit proteins range from 12,700 to 76,300 (average molecular weight: 28,700).

Inhibition of Respiration in Sea Urchin Spermatozoa following Interaction with Fixed Unfertilized Eggs VI. Probable Difference between the Species in the Mechanism for the Fixed-Egg-Induced Inhibition of Sperm Respiration: (respiration/sperm/sea urchin/carnitine/sperm-egg interaction).

In spermatozoa of all examined sea urchins, the respiration was inhibited and their motility was lowered by the glutaraldehyde-fixed eggs. The respiration of the fixed-egg-reacted spermatozoa was stimulated by 2, 4 dinitrophenol in Clypeaster japonicus, Hemicentrotus pulcherrimus and pseudocentrotus depressus but was not in Anthocidaris crassispina and Toxopneustes pileolus. Ratio of ADP to ATP was markedly lower in the reacted spermatozoa of the former species than in those of Anthocidaris. The low respiratory rate in the former species probably results from ADP control but does not in the latter species. Tetramethyl-p-phenylenediamine enhanced the respiratory rate in the reacted spermatozoa of the latter species to almost the same rate as in the intact spermatozoa, but elevated slightly in the former species. The inhibition of electron transport in mitochondrial respiratory chain is probably predominant in the latter species. In the former species, the slight inhibition of electron transport does not seem to result in a failure of ADP phosphorylation, and hence the stop of movement probably causes a shortage of ADP. Carnitine, which made the reacted spermatozoa of all species motile, enhanced the respiratory rate only in those of the former species.

Comparative studies on particulate acid phosphatases in sea urchin eggs

1. 1. Particulate acid phosphatases were partially purified and characterized in the eggs of six species of sea urchins and one species of sand dollar, Hemicentrotus pulcherrimus, Anthocidaris crassispina, Pseudocentrotus depressus, Strongylocentrotus purpuratus, S. francicanus, S. droebachiensis and Dendraster excentricus. 2. 2. The enzymes of 7 species were strongly inhibited by NaF, tartrate, p- chloromercuribenzoate and HgCl 2. 3. 3. Substrate specificity of the enzymes of all species was broad.

Recovery of sliding ability in arm-depleted flagellar axonemes after recombination with extracted dynein I.

We compared sliding velocity between outer doublet tubules in demembranated axonemes of sea-urchin (Pseudocentrotus depressus) sperm flagella with that of arm-depleted axonemes recombined with extracted dynein I. The outer arm-depleted axonemes after extraction with 0.5 M NaCl had a velocity of 6.9 +/- I.0 micrometer/s, while the intact axonemes had a velocity of 14.3 +/- I.5 micrometer/s in the presence of I mM ATP and 2 microgram/ml trypsin at 25 degrees C. The sliding velocity was closely related to the number of remaining outer arms following the NaCl-extraction process. When the outer arm-depleted axonemes were recombined with dynein I, the sliding velocity increased to 11.3 +/- 1.3 micrometer/s. Electron microscopy confirmed the recovery of 94% of outer arms in the axonemes. After extraction with Tris-EDTA solution for 10 min, the axonemes lost their sliding ability completely, even in the presence of ATP and trypsin. Such axonemes lacked most of both inner and outer arms, although sometimes the basal segment of the arms appeared to remain. When the exogenous dynein I fraction extracted from other axonemes was added, the axonemes could extrude tubules, and both types of arms reappeared clearly and distinctly in the axonemes. The recombined axonemes with one-fold stoichiometric excess of dynein I recovered 58% of the total number of arms and had a velocity of 7.4 +/- 1.6 micrometer/s. Those with 2-fold stoichiometric excess had a velocity of 11.0 +/- 1.5 micrometer/s, up to 82% of the arms in these axonemes being restored. These results indicated that the exogenous dynein I fraction derived from the outer arms restored sliding ability to arm-depleted axonemes, recombining with th outer doublet tubules as inner and outer arms, and that the sliding velocity had a close relationship to the total number of arms in the axonemes, irrespective of their being inner or outer arms.

Fibronectin from the ovary of the sea urchin,Pseudocentrotus depressus.

Fibronectin, with a subunit molecular weight of 220,000 daltons, was isolated from the ovary of the sea urchin,Pseudocentrotus depressus, using affinity chromatography on heat-denatured mammalian collagen coupled to Sepharose 4B. The distribution of fibronectin in the sea urchin ovary was examined by indirect immunofluorescence using antifibronectin serum. The basement membrane and the connective tissues exhibited strong fluorescence. The fibronectin was localized closely together with collagen bundles in the sea urchin ovary. Biochemical and immunological examinations indicate that sea urchin fibronectin has similar properties as those of mammalian fibronectin.

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWIGN INTERACTION WITH FIXED UNFERTILIZED EGGS: III. INHIBITION OF SPERM RESPIRATION BY HEAT STABLE SUBSTANCE REMOVED FROM GLUTARALDEHYDE-FIXED EGGS.

The respiratory rate of spermatozoa of the sea urchin, Pseudocentrotus depressus and Hemicentrotus pulcherrimus, became quite low and spermatozoa was immotile, after sperm suspension containing glutaraldehyde-fixed eggs of homologous species was stirred at 20°C for 15 min. The respiratory rate of fresh spermatozoa, introduced to the suspension of immotile spermatozoa thus obtained, was also reduced markedly. The respiration of fresh spermatozoa was not inhibited by adding them to suspension of intact or acrosome reacted spermatozoa. A heat stable and non-dialyzable substance, which inhibited sperm respiration, was removed from the fixed eggs by vigorously stirring the egg suspension for 10 min, when unfertilized eggs were fixed with insufficient amount of glutaraldehyde (10 ml of 1% glutaraldehyde solution to 1 ml egg pellet).

Sliding velocity between outer doublet microtubules of sea-urchin sperm axonemes.

Using a dark-field microscope equipped with a high-efficiency TV camera including a video tape-recorder, we recorded the sliding movement between outer doublet microtubules of the demembranated axonemes of sea-urchin (Pseudocentrotus depressus and Hemicentrotus pulcherrimus) sperm flagella by adding ATP and trypsin at 25 degrees C. The time and length of the sliding doublet microtubules from axonemes were measured directly from the image on the picture monitor from the video tape. The sliding velocity was almost constant in the range from 0 to 2% polyethylene glycol concentration in the reactivation medium and decreased a little at more than 2%. We prepared various lengths of axoneme fragments by homogenizing whole axonemes and found that the shorter fragments showed similar sliding velocity to that of longer ones at less than 200 microM ATP, but slightly decreased speed at more than 500 microM. ATP. The sliding movement sometimes stopped and the percentage of sliding axonemes was lower below 2 micrograms/ml trypsin. Above 3 micrograms/ml, the process appeared to be more like disintegration than sliding movement, which may be due to excess digestion by trypsin. Sliding speed was therefore measured in a reactivation medium containing 2% polyethylene glycol with the addition of ATP and 2 micrograms/ml trypsin. The velocity increased in proportion to the increase in ATP concentration. Vmax was approximately 14 micrograms/s at 2 mM ATP. In order to compare the Km for the sliding velocity with that of the ATPase activity of the axonemes, we measured ATPase activity of axonemes prepared and assayed under conditions in which sliding movement in the axonemes could be induced. Neither the curve of ATPase activity nor the curve of sliding velocity plotted against ATP concentration obeyed Michaelis-Menten kinetics. The close relationship between ATPase activity and sliding velocity suggested that 'sliding-movement-coupled ATPase activity' may well be reflected in the axoneme ATPase reported here.

RELATION BETWEEN THE ACROSOME REACTION AND FERTILIZATION IN THE SEA URCHIN. II. FERTILIZATION IN ACIDIFIED SEA WATER WITH EGG-WATER-TREATED SPERMATOZOA.

Fertilization of sea urchin eggs fails to occur at a pH lower than 6.5. Analytical studies on this problem were made with Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus. If the spermatozoa have been pretreated with egg water, eggs can be fertilized even at pH 6.5 and 6.0. The acrosome reaction is inhibited at a pH lower than 6.5. Intact spermatozoa fail to adhere to the fixed eggs in acidified sea water, whereas egg-water-treated spermatozoa adhere even at pH 6.5 and 6.0. From these results we infer that the failure of fertilization at pH 6.5-6.0 is caused by non-occurrence of the acrosome reaction, and that fertilization reactions other than the acrosome reaction, such as the binding and fusion of the gametes, are not inhibited in this range of pH. At pH 5.5, the spermatozoa become inert and fertilization is inhibited or suppressed, even though egg-water-treated spermatozoa are employed.

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWING INTERACTION WITH FIXED UNFERTILIZED EGGS.: I. CHANGE IN THE RESPIRATORY RATE OF SPERMATOZOA IN THE PRESENCE OF FIXED EGGS.

The respiratory rate of spermatozoa of the sea urchins, Anthocidaris crassispina, Clypeaster japonicus and Pseudocentrotus depressus, decreases markedly in the presence of homologous unfertilized eggs fixed with glutaraldehyde. No decrease in the rate of respiration occurs in the presence of fixed fertilized eggs. Fixed unfertilized eggs of different sea urchin species do not cause any change in the rate of sperm respiration. Spermatozoa adhere only to the fixed unfertilized eggs of the same species and are removed by a stirring for 5 min on a magnetic stirrer. The spermatozoa thus removed, are immotile and their respiratory rate is quite lower than that of motile spermatozoa in a control suspension stirred for 5 min. Intact spermatozoa adhere to the fixed eggs, from which the attached spermatozoa have been removed, and the respiratory rate of the spermatozoa also becomes quite low.

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWING INTERACTION WITH FIXED UNFERTILIZED EGGS: II. CAPACITY OF THE GLUTARALDEHYDE FIXED UNFERTILIZED EGGS FOR THE INHIBITION OF THE SPERM RESPIRATION.

Glutaradehyde-fixed unfertilized eggs of the sea urchin, Pseudocentrotus depressus, which bound spermatozoa, were capable of causing an inhibition of respiration in 2.3 ± 0.27 × 104 spermatozoa per egg. After stirring a suspension containing fixed eggs (2 × 103 -2 × 104 per ml) and a larger number of spematozoa than 2.5 × 104 per egg for 15 min at 20°c, the fixed eggs isolated from the suspension failed to cause any inhibition of respiration of fresh spermatozoa, and were incapable of binding fresh eggs. Fixed eggs, treated with a smaller number of spermatozoa than 2 × 104 per egg, had a capacity to bind spermatozoa and inhibited sperm respiration. Unless the fixed eggs lost the sperm-binding capacity, they exerted an inhibitory effect on the respiration of spermatozoa.

ATP-driven tubule extrusion from axonemes without outer dynein arms of sea-urchin sperm flagella.

We have prepared axonemes without outer dynein arms from sea-urchin (Pseudocentrotus depressus, Hemicentrotus pulcherrimus) sperm flagella by selective solubilization with NaCl. Electron microscopy revealed that the axonemes gradually lost their outer arms in 0.5 M NaCl during 10 min. Such axonemes retained 42.8 +/- 7.3% of their total axonemal ATPase activity and showed C, A, D and B bands in the dynein region of 4% SDS-gel, while a solubilized fraction of the outer arms consisted almost entirely of A band polypeptide. We have succeeded in causing extrusion of the outer doublets from such axonemes by addition of ATP and trypsin. A bundle of outer doublets was sometimes observed to be extruded first from an axoneme and to show bending motion for a while, subsequently followed by a sliding of separate doublets past each other. The speed of the tubule extrusion process was slower and around 60% of that of intact axonemes having both types of arm. These observations indicate that the inner arms have a function equivalent to that of the outer arms, of sliding on adjacent doublets, although the inner arms seem to be constituted from polypeptide(s) different from that of the outer arms.

Identification of the sialic acids from the egg jelly coat of the sea urchin Pseudocentrotus depressus (Okayama).

The jelly coat substance of the sea urchin eggs of Pseudocentrotus depressus (Okayama) was found to contain 0.35% sialic acids. After isolation of the sialic acids from the purified sulfated sialoglycoprotein containing 11% of sialic acids, analysis by colorimetry, thin-layer chromatography and gas-liquid chromatography/mass spectrometry demonstrated the nature of the sialic acids to be N-glycolylneuraminic acid and 9-O-acetyl-N-glycoloylneuraminic acid. The molar ratio between both sialic acids was found to be 1 : 1. Since it is known that O-acyl groups are partially released during the hydrolysis and isolation procedure, it can be assumed that in the native glycoprotein the percentage of O-acetylation is much higher than 50%. The sugar and sulfate composition of the glycoprotein is also described.

Conformational change in the outer doublet microtubules from sea urchin sperm flagella.

Dark-field microscopy with a high-powered light source revealed that the outer doublet microtubules (DMTs) from sea urchin (Pseudocentrotus depressus and Hemicentrotus pulcherrimus) sperm flagella assume helically coiled configurations (Miki-Noumura, T., and R. Kamiya. 1976. Exp. Cell Res. 97: 451.). We report here that the DMTs change shape when the pH or Ca-ion concentration is changed. The DMTs assumed a left-handed helical shape with a diameter of 3.7 +/- 0.5 micron and a pitch of 2.8 +/- 0.7 micron at pH 7.4 in the presence of 0.1 mM CaCl2, 1 mM MgSO4, and 10 mM Tris-HCl. When the pH was raised to 8.3, the helical diameter and pitch decreased to 2.1 +/- 0.1 micron and 1.3 +/- 0.3 micron, respectively. This transformation was a rapid and reversible process and was completed within 1 min. Between pH 7.2 and 8.3, the DMTs assumed intermediate shapes. When the Ca-ion concentration was depleted with EGTA, the helical structure became significantly larger in both pitch and diameter. For instance, the diameter was 3.8 +/- 0.4 micron at pH 8.3 in the presence of 1 mM EGTA and 2 mM MgSO4. Using a Ca-buffer system, we obtained results which suggested that this Ca-induced transformation took place at a Ca concentration of approximately 10(-7) M. These results were highly reproducible. The conformational changes in the DMT may play some role in the bending wave form of flagellar movement.

Possible causal relation between the acrosome reaction and cross‐fertilization in the sea urchins hemicentrotus pulcherrimus and pseudocentrotus depressus

AbstractSperm of Hemicentrotus pulcherrimus undergo the acrosome reaction in the jelly coat or on the surface of the vitelline layer of Pseudocentrotus depressus eggs and can fertilize the latter, whereas those of P depressus do not undergo the acrosome reaction even after they strike the vitelline layer of H pulcherrimus eggs and cannot cross‐fertilize.In the latter combination, however, if cross‐insemination is performed in the presence of homologous egg water, cross‐fertilization becomes less difficult than in normal seawater, and percentage cross‐fertilization rises as does percentage acrosome reaction. It is suggested that the cross‐fertilizability of this combination depends highly on the inductivity of the acrosome reaction. The reason why such a causal relation is observed is discussed.

Two pathways from glycogen to glucose‐6‐phosphate in sea urchin eggs with special reference to the difference between the species in the contribution ratio of each pathway

AbstractIn the unfertilized eggs of the sea urchins, Anthocidaris crassispina, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, Mespilia globulus, Temnopleurus toreumaticus, Toxopeneustes pileolus, and Clypeaster japonicus, the activities of phosphorylase [EC 2.4.1.1], phosphoglucomutase [EC 2.7.5.1], exo‐l,4‐α‐glucosidase [EC 3.2.1.3], and hexokinase [EC 2.7.1.1] are very similar. In all species, only phosphorylase activity is higher in fertilized eggs than in unfertilized eggs. The concentrations of glycogen, glucose, GIP, G6P, ATP, ADP, and Pi; the products and substrates in reactions catalyzed by these enzymes, were measured in these eggs. Based on the concentrations of these compounds in the eggs, it is assumed that G6P is produced by the combined action of glucosidase and hexokinase in all species examined, and that it is also produced in the reaction catalyzed by phosphorylase and phosphoglucomutase in all species except A crassispina and P depressus. Glycogen was found both in supernatant and in precipitate fractions, which were obtained by adding perchloric acid. Glycogen in the precipitate seems to be protein‐bound. Whole glycogen level in the eggs is almost the same in all species examined, but the level of acid‐soluble glycogen, as well as GIP, is markedly lower in the eggs of A crassispina and P depressus than in the eggs of other species examined. Protein‐bound glycogen is utilized by glucosidase activity but not by phosphorylase activity, in contrast to acid‐soluble glycogen, which is utilized by both enzyme activities. Hence, it is assumed that the failure of G6P production by phosphorylase and phosphoglucomutase‐in A crassispina and P depressus eggs is due to a low level of acid‐soluble glycogen in these eggs.

CHANGES IN ACTIVITIES OF CREATINE KINASE, ARGININE KINASE AND THEIR MULTIENZYME FORMS DURING EMBRYONIC AND LARVAL DEVELOPMENT OF SEA URCHINS.

The presence and the changes of CPK and APK have been studied during larval development through metamorphosing of Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudo-centrotus depressus. While no CPK activity was found in the unfertilized eggs and the embryos of early developmental stages, APK was quite active throughout these stages. At the late 8-armed pluteus stage just prior to metamorphosis, CPK first became active. Electrophoretically this CPK was identical with one of three CPK forms of sperm, tube feet and esophagus but not with two CPK forms of lantern muscle. APK in the unfertilized eggs and early embryos was electrophoretically separated into two distinct molecular forms, one of which disappeared during the late larval stages. The persisting form of larval APK was identical with a single APK form present in the adult muscular tissues.

When do sperm of the sea urchin, Pseudocentrotus depressus, undergo the acrosome reaction at fertilization?

Jelly coats of the sea urchin, Pseudocentrotus depressus, were stripped off the eggs, and the eggs were “inseminated.” After penetration through the isolated jelly hull, sperm swarmed in the cavity previously occupied by the egg. Electron microscopic examination could not detect any sperm with reacted acrosome. Observation was also made of the sperm penetrating through the intact jelly coat-egg complex. Although a number of sperm were examined in ultrathin sections, only those attached to the vitelline layer had undergone the acrosome reaction; those sperm embedded in jelly but not attached to the vitelline layer had not undergone the acrosome reaction. The sequence of events in fertilization of this species and of other echinoids is discussed.

EXOGASTRULATION INDUCED BY CHILLING IN SEA URCHIN LARVAE.

Exogastrulation is induced by chilling in several species of sea urchins, including Strongylocentrotus intermedius, Strongylocentrotus nudus, Pseudocentrotus depressus and Anthocidaris crassispina, but not in Hemicentrotus pulcherrimus. When early gastrulae are raised at low temperatures, no pseudopodia of secondary mesenchyme cells are formed, but the invagination of the endodermal plate occurs normally. When gastrulae in later stage having pseudopodia are chilled, the pseudopodia withdraw and the archenteron begins to retract, resulting in exogastrulation. The exogastrulae are also induced when the larvae are raised in the presence of colchicine, vinblastine, cytochalasin B or cytochalasin C. The formation of exogastrula induced by chilling is presumed to be caused by the depolymerization of microtubules in the secondary mesenchyme cells and their pseudopodia. The fully invaginated archenteron of the late gastrula, even when it is chilled, remains within the blastocoel and does not evaginate. The effectiveness of low temperature treatment in inducing exogastrulation may be related to the environmental temperature at the breeding season of the animal.