Pseudocentrotus Depressus Research Articles

Isolation and Properties of a New Type of Sialopolysaccharide-Protein Complex from the Jelly Coat of Sea Urchin Eggs

Abstract A heteropolysaccharide-protein complex containing 70% sialic acid was isolated from the jelly coat of the eggs of the sea urchin, Pseudocentrotus depressus. The yield of this sialopolysaccharide-protein complex was 0.32% of the whole jelly powder. Purified by centrifugation and chromatography on Bio-Gel, the complex was found to be homogeneous by the criteria of electrophoresis and ultracentrifugation. Its sedimentation coefficient, s020 ,w, was 5.1, and the material carried a strong negative charge. The carbohydrate composition of the complex, expressed in percentages of the dry weight, was sialic acid, 70.0; fucose, 9.2; hexose, 3.7; and hexosamine, 2.4. Analyses for glycolic acid strongly indicated that all of the sialic acid occurred in glycolyl form. The molar ratio of sulfate to fucose was 1.42:1. The combined weights of monosaccharide, sulfate, and amino acid residues accounted for 96.0% of the complex weight. Thin layer chromatography of the sialopolysaccharide-protein complex after hydrolysis in mild acid showed five spots stained by Ehrlich's reagent. The major spot migrated as N-glycolylneuraminic acid. No indication of N-acetylneuraminic acid appeared. It is proposed that there are several new, undescribed neuraminic acids in the jelly coat, since the sialic acid yielded only 45% of the color in the reaction with thiobarbituric acid as compared with the direct Ehrlich assay and, moreover, no release of sialic acid from the complex by neuraminidase treatment was obtained.

Polymerization of actin from sea urchin eggs

Actin was isolated and purified from unfertilized sea urchin eggs ( Pseudocentrotus depressus and Hemicentrotus pulcherrimus), by a method which utilizes its specific binding activity with myosin from rabbit skeletal muscle. The purified actin exists as a monomer (ovo-G-actin) in salt-free solution. The ovo-G-actin has a sedimentation coefficient of 3.0–3.2 S. On the addition of salt (0.1 M KCl), the ovo-G-actin transforms into a heavy component of 25–28 S, which has a higher viscosity value ( η sp/c) of 0.9 – 2.1 dl/g. The heavy component (ovo-F-actin) was revealed by electron microscopy to be a filament having a diameter of 60–80 Å. The ovo-G-actin in salt-free solution also transforms into the ovo-F-actin on the addition of divalent salts, CaCl 2 or MgCl 2.

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.

A study on the possible significance of carbohydrate moiety in the sperm-binding protein from sea urchin egg

A possible significance of carbohydrate moiety in sperm-binding protein of eggs of the sea urchins, Hemicentrotus pulcherrimus and Pseudocentrotus depressus, was examined by the following two experiments. 1. 1. Glycopeptide probably having original carbohydrate moiety was obtained by trypsin-digestion and gel-filtration. It was not functional in sperm-binding at all. 2. 2. Carbohydrate moiety of the original material was oxidized by periodate. Such a protein was fully functional in sperm-binding. It was suggested that the carbohydrate moiety is not essential in the sperm-binding.

Pathways of carbohydrate breakdown in sea urchin eggs

The relative participation of the pentose phosphate cycle to over-all carbohydrate metabolism has been estimated from the amounts of 14CO 2 produced from 14C-1- and 14C-6-glucose, in eggs and embryos of the sea urchin, Pseudocentrotus depressus. It has been shown that a greater part of glucose is oxidized via the pentose phosphate cycle during development up to the gastrula stage, after which glucose is degraded mainly through glycolysis and tricarboxylic acid cycle. An enhancement of the pentose-cycle activity occurs at fertilization and during cleavage stages.

Cell division: direct measurement of maximum tension exerted by furrow of echinoderm eggs.

Tensions exerted by cleavage furrows in isometric contraction were measured by means of flexible glass needles whose characteristics of bending had already been determined. The tension of Astriclypeus manni furrows in second division is 3.04 x 10(-3) +/- 0.95 x 10(-3) dyne; that for Pseudocentrotus depressus eggs in first division is 2.00 X 10(-3) +/- 0.43 x 10(-3) dyne. The tension required for cleavage probably does not exceed 1.5 x 10(-3) dyne. According to existing morphological evidence, these values can be accounted for by a substance whose capacity for exerting tension does not exceed that of an actomyosin thread.

Accumulation of radioactive calcium in larvae of the sea urchin Pseudocentrotus depressus: Nakano, E., K. Okazaki and T. Iwamatsu, 1963. Biol. Bull., 125 (1): 125–132

This study examined how contraction force and protein profiles of the tube feet of the sea urchin (Pseudocentrotus depressus) were affected when acclimated to 400 (control), 2000 and 10,000 μatm CO2 for 48 days. Acclimation to higher CO2 conditions significantly reduced contraction force of the tube feet. Two-dimensional gel electrophoresis showed that eight spots changed in protein volume: six up-regulated and two down-regulated. Using matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight mass spectrometry, three up-regulated spots (tubulin beta chain, tropomyosin fragment, and actin N-terminal fragment) and two down-regulated spots (actin C-terminal fragment and myosin light chain) were identified. One possible interpretation of the results is that elevated CO2 weakened contraction of the tube feet muscle through an alteration of proteome composition, mainly associated with post-translational processing/proteolysis of muscle-related proteins.

Release of acid polysaccharides following fertilization of sea urchin eggs: A chemical study of changes in the cell surface

1. 1. At the time of fertilization Japanese sea urchin eggs released organic substances into the surrounding sea water, which reduced KMnO 4 and were non-dialyzable, thermostable, and alcohol-insoluble. 2. 2. The fertilization product of the Pseudocentrotus depressus egg contained acid polysaccharides composed of glucose, galactose, mannose, hexosamine, sulfate and probably also amino acid. The fertilization product of the Anthocidaris crassispina egg contained acid polysaccharides composed of the same constituents except for glucose and mannose. 3. 3. No release of free sulfates from the eggs of the above two species could be demonstrated. 4. 4. Fifty to 100 per cent of the amount of fertilization product appeared in the suspension medium during the first 5 min after insemination, the remainder appearing within 5 to 10 min after insemination. The normal eggs retained the same substance in perivitelline space formed at fertilization. 5. 5. The pH of the surrounding sea water markedly decreased shortly after fertilization in unbuffered artificial sea water; the drop in pH of the suspension medium of trypsin-treated eggs and urea-treated eggs was not as great as that of the suspension medium with intact egg, suggesting that other compounds with buffering action may also have been released by the treated eggs. 6. 6. It is highly probable that the release of the fertilization product corresponds, in part at least, to the acid formation occurring very soon after fertilization.

STUDIES ON THE RESPIRATION OF SEA-URCHIN SPERMATOZOA. V. THE EFFECT OF P-C02.

ABSTRACT The effect of PCO2 on the respiration and motility of sea-urchin spermatozoa was studied on Anthoddaris crassispina. Some points were also corroborated on Hemicentrotus pulcherrimus, Pseudocentrotus depressus, Paracentrotus lividus and Sphaerechinus granularis. It was found that any level of CO2 above 1 %, both in oxygen and in air, inhibited the O2 uptake of spermatozoa suspended in sea water, measured polarographically with a vibrating platinum electrode. The inhibitory effect paralleled the PCO2and was completely reversed by introducing oxygen or air. pH variations between 8·50 and 6·75 had no influence on O2 uptake, when the pH was stabilized with 0·05 Mhistidine-HCl-NaOH. O2 uptake was, however, reduced to some extent outside this range, especially on the acid side. Although the increase in PCO2 is inevitably followed by a decrease in pH, the inhibitory effect of CO2 far exceeds that caused by the reduction in pH. The O2 uptake rate was little affected by the addition of both bicarbonate and carbonate ions to the suspending medium, although the former had a slightly stimulating effect at certain concentrations. In buffered sea water, CO2 had little influence on O2 uptake even at partial pressures as high as 10% which inhibited the bulk of O2 uptake in sea water. Sperm motility was also inhibited by CO2. In this case, too, the inhibition paralleled the PCO2 and was completely reversible. The effect was more pronounced in air than in oxygen, and in dense sperm suspensions than in dilute ones. These results suggest that gaseous CO2 is the factor responsible for the inhibitory effect. The possible role of CO2 in the dilution phenomena of sea-urchin spermatozoa is discussed.

RESPIRATORY LEVEL OF SEA URCHIN EGGS BEFORE AND AFTER FERTILIZATION

The respiratory level of sea urchin eggs was measured before and after fertilization, using three Japanese species, Pseudocentrotus depressus, Hemicentrotus pulcherrimus and Anthocidaris crassispina. The rate of oxygen uptake of unfertilized eggs is low even immediately after shedding, and it remains constant for more than three hours. Upon fertilization, the rate of oxygen uptake of the eggs increases sharply, and the increase may occur under natural conditions.

Changes in the content of adenine nucleotides upon aerobic incubation of sea urchin spermatozoa

1. 1. The content of adenine nucleotides in spermatozoa of the sea urchins, Pseudocentrotus depressus and Hemicentrotus pulcherrimus, was determined by a column chromatographic method. 2. 2. In Pseudocentrotus spermatozoa, the proportion of AMP was highest and that of ATP lowest, while in Hemicentrotus spermatozoa, the content of ADP was somewhat higher than that of AMP. 3. 3. Under the conditions that allowed extensive exhaustion of endogenous substrates, a significant decrease in the content of ADP and ATP was observed on aerobic incubation of sperm suspensions of both species. 4. 4. When, conversely, the spermatozoa were placed under conditions where utilization of endogenous substrates was low, the content of ADP and ATP did not show any marked change even after prolonged incubation.

Studies On the Respiration of Sea-Urchin Spermatozoa

ABSTRACT The respiratory quotient of sea-urchin spermatozoa has been determined with Pseudocentrotus depressus, Anthocidaris crassispina and Hemicentrotus pulcherrimus. The R.Q. of sea-urchin spermatozoa, measured by the Warburg direct method, has been reported to be near unity. This was also the case with the present material when the suspending medium was sea water, the R.Q. being 0·8–1·0. It was found, however, that the pH of sperm suspensions was markedly different in the presence and absence of alkali to absorb CO2. When the pH of the suspension was fixed by such buffers as 0·025 M-glycyl glycine, the R.Q. measured by the above method was about 0·7. This is in accord with the results of earlier metabolic studies, which indicated that endogenous phospholipids are the main substrates for the respiration of sea-urchin spermatozoa. The O2 uptake of the present material, however, was found to be little affected by variation in pH. The difference in the R.Q. values obtained with ordinary sea water and buffered sea water, therefore, cannot be explained in terms of pH. When the spermatozoa were suspended in ordinary sea water, the utilization of endogenous phospholipids was much reduced in the absence of alkali, while in buffered sea water the change in phospholipids was almost the same, with and without the absorption of CO2. Determination of the R.Q. by the first method of Dickens & Šimer, in which the O2 uptake and the CO2 output were measured with one and the same sperm suspension, gave a value of about 0·7 with both ordinary and buffered sea water.

Studies on sulfhydryl groups during cell division of sea urchin egg: I. Glutathione

1. 1. The amounts of reduced and oxidized glutathione during early cell division of the sea urchin egg were measured. 2. 2. TCA-soluble SH groups were found to fluctuate in correlation with the cell division as in Rapkine's work. 3. 3. In TCA-soluble SH groups, glutathione and protein are contained side by side, and after the removal of the protein, changes in the amounts of SH groups in relation to cell division disappear. 4. 4. Constant residual SH values after deproteinization are as follows: GSH × 10 −s8 mg/egg GSSG × 10 −s8 mg/egg Pseudocentrotus depressus 71 6 Hemicentroius pulcherrimus 60 Clypeaster japonicus 102 7

A cytochemical study of the sulfhydryl groups of sea urchin eggs during the first cleavage.

In the eggs of four species of echinoderms, Mespilia globulus, Pseudocentrotus depressus, Hemicentrotus pulcherrimus and Clypeaster japonicus, changes in the distribution of protein-bound SH groups from fertilization to the 2 cell stage have been studied cytochemically by use of a mercaptide-forming azo dye. In the eggs of these species, the color intensity in the cytoplasm increased upon fertilization. The astral centers and spindle during mitosis were stained deeply. When the aster formation was suppressed by ether, hyaline spots appeared in the egg cytoplasm instead of well formed astral centers and these spots were stained by the SH-specific dye. Upon recovery of such eggs in pure sea water, and when cleavage ensued, such spots disappeared and two new astral centers were reorganized. The SH-protein occurring in the centrosphere is considered to be the precursor material for the asters and spindle, and this material is apparently derived from the cytoplasm.

On the possible role of high energy phosphate in the cortical change of sea urchin eggs: I. Effect of dinitrophenol and sodium azide

1. 1. Treatment of unfertilized eggs of the sea urchins, Heliocidaris crassispina and Temnopleurus toreumaticus with DNP and NaN 3 prior to insemination was found to inhibit breakdown of the cortical granules, elevation of the fertilization membrane and the sperm block at the time of fertilization. This inhibition of the cortical change by the uncouplers is reversible. 2. 2. Essentially the same inhibitory effect was observed on the cortical change parthenogenetically induced by butyric acid or urea. 3. 3. The uncouplers, however, do not block the cortical change induced by Monogen or Na-choleinate. 4. 4. In Pseudocentrotus depressus, when the pretreatment is carried out at lower temperatures (14–20 °C), neither DNP nor NaN 3 is inhibitory; but at higher temperatures (27–29 °C), their inhibitory effect on the cortical change is always apparent. Moreover, when eggs are subjected to these uncouplers at a higher temperature for a sufficient time-period to produce almost complete inhibition, and are thereafter placed into sea water of lower temperature containing the uncouplers, a reversal of the inhibition takes place. 5. 5. Addition of ATP to DNP-sea water does not remove the inhibition produced by DNP. 6. 6. It is suggested that the uncouplers primarily act on some of the early steps of the chain of reactions making up the cortical change, presumably on the fertilization wave, in which high energy phosphate may participate.

CYTO-EMBRYOLOGICAL STUDIES OF SEA URCHINS. III. ROLE OF THE SECONDARY MESENCHYME CELLS IN THE FORMATION OF THE PRIMITIVE GUT IN SEA URCHIN LARVAE

1. The first half of the gastrulation process in sea urchins begins with an in toto invagination of the endodermal plate, followed by its stretching.2. In the second half of the process, pseudopodia are sent out by the secondary mesenchyme cells toward the animal pole. These pseudopodia attach to the blastular wall and pull the archenteron up toward that pole.3. The pulling capacity of the pseudopodia is shown by the displacement toward the secondary mesenchyme cells of oil droplets injected in the blastocoel.4. When the pseudopodial connection is artificially severed by one of the following methods, exogastrulae result:a) Blastocoelic expansion after sucrose treatment (Mespilia globulus, Clypeaster japonicus).b) Ca-low treatment (Clypeaster, Pseudocentrotus depressus).c) Pancreatin treatment (Mespilia).5. Abnormal invagination in various degrees brought about by the above treatments can be accounted for by differences in their effects on the pseudopodia formation.6. Pseudopodia of the secondary mesenchym...

Exogastrulation and Differential Cell Dissociation by Sodium Azide in Dendraster excentricus and Patiria miniata

Previous articleNext article No AccessExogastrulation and Differential Cell Dissociation by Sodium Azide in Dendraster excentricus and Patiria miniataC. M. ChildC. M. Child Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by Volume 26, Number 1Jan., 1953 Article DOIhttps://doi.org/10.1086/physzool.26.1.30152149 Views: 2Total views on this site Citations: 2Citations are reported from Crossref Journal History This article was published in Physiological Zoology (1928-1998), which is continued by Physiological and Biochemical Zoology (1999-present). Copyright 1953 The University of Chicago PressPDF download Crossref reports the following articles citing this article:Matilda Moldenhauer Brooks The electrical property of matter, the trigger mechanism controlling cell growth, Protoplasma 57, no.1-41-4 (Mar 1963): 144–157.https://doi.org/10.1007/BF01252050KAYO OKAZAKI EXOGASTRULATION INDUCED BY CALCIUM DEFICIENCY IN THE SEA URCHIN, PSEUDOCENTROTUS DEPRESSUS *, Embryologia 3, no.11 (Jan 1956): 23–36.https://doi.org/10.1111/j.1440-169X.1956.tb00058.x

The role of the cortical granules in the formation of the fertilization membrane in eggs from Japanese sea urchins. I

1. 1. Strongylocentrotus pulcherrimus, Anthocidaris crassipina, Mespilia globulus, Pseudocentrotus depressus, Clypeaster japonicus, and Peronella lesueuri were examined concerning the behavior of the cortical granules in the formation of the fertilization membrane. 2. 2. In the eggs of Clypeaster japonicus, the cortical granules are differentiated into two optical parts of light and dark, when observed by a phase contrast microscope (dark contrast). 3. 3. Following the attachment of a spermatozoon, the granules explode and change into large globules. The granule explosion begins from the attachment point of the spermatozoon and spreads in a wave-like fashion. 4. 4. With this change, the globules are ejected, and the vitelline membrane is separated from the egg surface. 5. 5. The globules adhere to the rising vitelline membrane, gradually combining with it and giving rise to the fertilization membrane. 6. 6. When the vitelline membrane is weakened by proteolytic enzymes, the globules are squeezed out through the membrane, which remains attached to the egg surface. After several minutes, rod-like precipitates are formed in the globules. 7. 7. Ca-ion is necessary for the hardening of the fertilization membrane and also for the formation of the “rods.” 8. 8. It is suggested that the substance responsible for the hardening of the fertilization membrane is the same as the substance of the rods. 9. 9. The hatching enzyme of Strongylocentrotus pulcherrimus acts specifically on this substance.

RE-FERTILIZATION OF THE FERTILIZED EGGS OF THE SEA URCHIN

1. If the fertilized eggs of sea urchins (Strongylocentrotus pulcherrimus, Pseudocentrotus depressus, Heliocidaris crassispina, Temnopleurus toreumaticus) are inseminated again after removal of the fertilization membrane and washing with a Ca-Mg-free medium for a few minutes, re-fertilization can take place. Spermatozoa not only penetrate into the previously fertilized eggs but also take part in the formation of the mitotic figure, so that the cleavage of re-fertilized eggs is irregular and characteristic of polyspermy.2. Re-fertilization can occur without removing the membrane mechanically if the eggs have been treated with a Ca-Mg-free medium immediately after the first insemination.3. Re-fertilization can take place at any stage of the first cell division and even at the 2-cell stage.4. It is justifiable to consider that there is a sperm-excluding substance on the surface of fertilized eggs. This substance is stable in sea water but is easily lost in Ca-Mg-free media.

Sperm entrance in echinoderms; observed with the phase contrast microscope.

1. Phase contrast microscope observation of sperm penetration into the eggs of five species of sea urchins—Mespilia globulus, Clypeaster japonica, Strongylocentrotus pulcherrimus, Pseudocentrotus depressus, Heliocidaris crassispina—and one starfish—Asterina pectinifera—have shown that total engulfment of the spermatozoan including all of the tail, is the rule in these species.2. In every case the fertilization cone makes its first appearance after contact has been established between the sperm head and the living egg cytoplasm, and persists in normal cases for from 5 to 15 minutes after the beginning of sperm penetration. Subjection of the eggs to some experimental conditions—heat, cold, nicotine solution—causes abnormal production and persistence of the fertilization cones.3. Engulfment of the sperm tail in these species is usually complete within 2-4 minutes after the beginning of penetration. No significant change in engulfment time could be induced by exposing the eggs to the abnormal physiological conditions tested—heat, cold, aging, exposure to polyspermy-producing agents.4. In the two species with the most transparent eggs (Mespilia and Clypeaster), the movements of the sperm head within the cytoplasm can be clearly observed, from the time it passes the cortex until it becomes quiescent, which approximately coincides with the final engulfment of the tail. Progress of the spermatozoan appears to be effected by a combination of autonomous movement and streaming of the egg cytoplasm.5. About four minutes after the beginning of fertilization, the clear outline of the sperm head is lost and the sperm aster begins to develop. From this time the middle piece of the spermatozoan can be found lying in the cytoplasm, and traced as it moves together with the sperm aster toward the center of the egg. So far as could be determined, it takes no active part in the first cleavage.6. In one case a sperm head, which had entered an egg close to the exceptionally excentric egg pronucleus, was attracted to and held motionless against the pronuclear membrane, within about a minute and a half after entering the cytoplasm, while its tail was still largely outside the egg. This, however, produced no variation in the subsequent process of engulfment of the tail and formation of the sperm aster.