Ultrastructure of collagen in sea urchin embryos

Summary Collagen fibrils with a main period banding of 610 Å and 220 Å in width were observed in the blastocoel of 72-h embryos of the sea urchin,Strongylocentrotus purpuratus. Non-striated fibrils of 50 Å diameter were also observed. The collagen is seen in highest concentration in the vicinity of mesenchyme cells which are richly endowed with endoplasmic reticulum and secretory vesicles. A role for collagen in cell attachment, orientation and spicule formation is discussed.     

Development of the esophageal muscles in embryos of the sea urchin Strongylocentrotus purpuratus

Summary Development of the esophageal muscles in embryonic sea urchins is described using light- and electron microscopy. The muscles develop from processes of about 14 cells of the coelomic epithelium that become immunore-active to anti-actin at about 60 h (12–14° C). Initially, eachmyoblast extends a single process with numerous fine filopodia around the esophagus. By 72 h the processes have reached the midline and fused with those from cells of the contralateral coelomic sac. Myoblasts begin to migrate out of the coelomic epithelium between 72 and 84 h. By 72 h the processes stain with the F-actin specific probe NBD-phallacidin. The contractile apparatus is not evident in transmission electron-microscopic preparations of embryos at 70 h, but by 84 h the contractile apparatus is present and the muscle cells are capable of contraction. Because the myoblasts migrate free of the coelomic epithelium and are situated on the blastocoelar side of the basal lamina, it is suggested that that they should be considered as a class of mesenchymal cells.     

Cyclic appearance of cytoplasmic NOR-silver-stained particles in sea urchin embryos.

When sea urchin embryos were subjected to nucleolar organizer region (NOR)-silver staining, densely stained particles were observed in the cytoplasm. The appearance of these cytoplasmic particles (CPs) was cell-cycle dependent. During early development, the CPs were detected at interphase, but not during mitosis; they disappeared at metaphase and reappeared at telophase. The CPs appeared periodically even when embryos were treated with cytochalasin B or aphidicolin, which inhibits the progression of cytokinesis and nuclear division, respectively. By contrast, CPs were not detected in the colchicine-treated embryos in which both cytokinesis and nuclear divisions were prevented. The CPs were observed only in the embryos whose stage was early blastula (about 6th to 7th cleavage) or earlier; no CPs were detected even at interphase in the embryos at late blastula (about 8th to 9th cleavage) or later. Electron microscopic evaluation showed CPs to be granular structures, similar to heavy bodies. Also, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) showed that 95-kDa and 38-kDa proteins were the NOR-silver-staining proteins in sea urchin embryos. These proteins existed during the course of the cell cycles. These results suggest that (1) the cyclic appearance of the CPs or heavy bodies is closely related to the cell cycle as well as the programming of the embryogenesis, but independent of the cycle of cytokinesis and nuclear division; (2) 95-kDa and 38-kDa proteins are the major NOR-silver-staining proteins in sea urchin embryos.     

Apoptosis in early development of the sea urchin, Strongylocentrotus purpuratus

Apoptosis provides metazoans remarkable developmental flexibility by (1) eliminating damaged undifferentiated cells early in development and then (2) sculpting, patterning, and restructuring tissues during successive stages thereafter. We show here that apoptotic programmed cell death is infrequent and not obligatory during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus. During the first 30 h of urchin development, fewer than 20% of embryos exhibit any cell death. Cell death during the cleavage stages consists of necrotic or pathological cell death, while cell death during the blastula and gastrula stages is random and predominantly caspase-mediated apoptosis. Apoptosis remains infrequent during the late blastula stage followed by a gradual increase in frequency during gastrulation. Even after prolonged exposure during the cleavage period to chemical stress, apoptosis occurs in less than 50% of embryos and always around the pre-hatching stage. Embryonic suppression of apoptosis through caspase inhibition leads to functionally normal larvae that can survive to metamorphosis, but in the presence of inducers of apoptosis, caspase inhibition leads to deformed larvae and reduced survival. Remarkably, however, pharmacological induction of apoptosis, while reducing overall survival, also significantly accelerates development of the survivors such that metamorphosis occurs up to a week before controls.     

Cryopreservation of sea urchin embryos (Paracentrotus lividus) applied to marine ecotoxicological studies

Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me₂SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to −12 °C at 1 °C/min, holding for 2 min for seeding, cooling to −20 °C at 0.5 °C/min, and then cooling to −35 °C at 1 °C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.     

Commitment to vegetalized development in sea urchin embryos

Summary Strongylocentrotus purpuratus embryos were reared in 0.025 M LiCl, which causes commitment to vegetalized development within 5 h after treatment begun at fertilization. Treated and control embryos were labelled with³⁵S-methionine for 3 h intervals from 2–14 h, solubilized, and subjected to 2-dimensional polyacrylamide gel electrophoresis. Comparison of autoradiographs of the gels, in which over 400 proteins can be detected, indicate that while LiCl treatment causes a short delay in the initiation or cessation of synthesis of a few proteins, no qualitative or major quantitative differences can be detected between control and treated embryos. Normal gastrulae and vegetalized exogastrulae labelled 38 h after fertilization have several differences in patterns of protein synthesis. We conclude that the early determinative events involved in vegetalization are not reflected in detectable differences in the pattern of protein synthesis.     

Temperature limits to early development of the New Zealand sea urchin Evechinus chloroticus (Valenciennes, 1846)

Seawater temperature is an important environmental factor for the early life stages of marine invertebrates. In this study we evaluated and described the effects of temperature during early development of E. chloroticus, identifying the optimum temperature range and upper thermal limit for successful development. The temperature range evaluated was between 15–24 °C which included the normal seawater temperatures during the spawning season in northern New Zealand, as well as the highest temperature projected by the IPCC for this region due to global warming (1–3 °C by the year 2100). Gametes from several females and males were used in the experiment. Fertilization was carried out at different temperatures and development was monitored at different time points after fertilization in each temperature. The development rate of E. chloroticus increased with an increase in seawater temperature. However, at temperatures higher than 21.5 °C the amount of abnormal development reached ∼30%. The optimum temperature for early development was between 15–21 °C, whereas the upper thermal limit was ∼24 °C. Therefore, early development of E. chloroticus is negatively affected by an increase in seawater temperature of ∼3–4 °C above current seawater temperature levels in northern New Zealand. The thermal sensitivity of early life stages of E. chloroticus could affect survival rates during early development of this species in a global warming scenario, which could impair recruitment in populations which are exposed to higher temperatures, leading to possible distributional shifts of this species.     

dbEST-derived SSR markers in sea urchin (Hemicentrotus pulcherrimus)

Sea urchin is an important model organism for evolutionary biology, embryology, and gene regulation study. We developed and evaluated simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) of Strongylocentrotus purpuratus and Hemicentrotus pulcherrimus. Characteristics of nine EST-SSR loci were investigated using 41 Hemicentrotus pulcherrimus individuals. The number of alleles per locus ranged from two to five. The observed heterozygosity ranged from 0.122 to 0.7805, while the expected heterozygosity ranged from 0.4472 to 0.7696. These loci and markers will be useful for population genetics and systemic evolution among species of sea urchin.     

Electron microscope visualization of giant polysomes in sea urchin embryos

Chromatin spreading techniques have been applied to the electron microscopic visualization of polysomes in sea urchin (Strongylocentrotus purpuratus) eggs and embryos. Polysomes of giant size are commonly found after the 8-cell stage. The largest seen, from an early gastrula, was 13.6 m in length, carried 277 ribosomes, with a message calculated to contain 6.49×10⁴ nucleotides and potentially to encoded 2.38×10⁶ daltons of peptide. Polysomes are rare and very large ones absent from lysates of unfertilized eggs. Giant polysomes appear in 4- to 8-cell stages and are common in 16-cell stages and thereafter. They are of two forms: a compact form with no spacing between ribosomes characteristic of stages through early mesenchyme blastulae, and an extended form found only after late mesenchyme blastulae. Both have potential for massive informational content. Some of each type have ribosome-free tails at one end, as long as 733 Å in the compact forms, and 7,890 Å in the extended ones. Occasionally they have a single array of fibrous material increasing from one end of a polysome to the other, interpreted to be nascent peptide chains. Polysomes are not found after brief, mild exposure of lysates to RNase A, or from embryos treated with puromycin. Very large polysomes are present in lysates of blastulae exposed since fertilization to actinomycin D, cycloheximide, or cordycepin. They appear in parthenogenetically activated or fertilized enucleate merogones, but are absent from unactivated merogones, demonstrating that egg masked messages can generate them. A potential embryological significance of giant, potentially polycistronic polysomes is suggested.     

Inhibition of spicule elongation in sea urchin embryos by the acetylcholinesterase inhibitor eserine

The activity of acetylcholinesterase (AchE) increases rapidly after the gastrula stage of sea urchin development. In this report, changes in activity and in the molecular differentiation of AchE were investigated. AchE activity increased slightly during gastrulation and rose sharply thereafter, and was dependent on new RNA synthesis. No activity of butyrylcholinesterase was found. Morphogenesis in sea urchin embryos was inhibited by the AchE inhibitor eserine, which specifically inhibited arm rod formation but not body rod formation. Spicule formation and enzyme activity in cultured micromeres were inhibited by eserine in a dose-dependent manner. During gastrulation, two molecular forms of AchE were detected with polyacrylamide gel electrophoresis. The appearance of an additional band on the gel was consistent with the occurrence of a remarkable increase in the enzyme activity. This additional band appeared as a larger molecular form in Anthocidaris crassispina, Hemicentrotus pulcherrimus, Stomopneustes variolaris, and Strongylocentrotus nudus, and as a smaller form in Clypeaster japonicus and Temnopleurus hardwicki. These results suggest that the change in the molecular form of AchE induced a change in enzymatic activity that in turn may play a role in spicule elongation in sea urchin embryos.     

The contractility of elongated microvilli in early sea urchin embryos

Summary Elongated microvilli attach the early sea urchin embryo to the fertilization envelope and support it in a concentric position within the perivitelline space. The contractility of the elongated microvilli was demonstrated in several ways. (1) During normal cleavage, these microvilli change their length to adapt to the change in shape and numbers of blastomeres. (2) When treated with calcium-free sea water, embryos become eccentrically located and the microvilli extend further than normal on one side; when returned to normal sea water, the embryos become centered again. (3) Several agents cause the fertilization envelope to become higher and thinner than normal and the elongated microvilli to extend correspondingly if treated within ten min after fertilization. In some cases, both elongated microvilli and fertilization envelope return to normal size when returned to normal sea water. (4) Fertilization in a papain solution causes the elongated microvilli and the fertilization envelope to contract to the surface of the embryo. (5) Refertilization after the papain-induced contraction can bring about the elongation of these microvilli and the elevation of the fertilization envelope a second time. It was also shown that elongated microvilli are extended immediately upon fertilization, at the same time as the short microvilli. The firm adherence of the tips of elongated microvilli to the fertilization envelope by means of extracellular matrix fibers is shown in a high voltage electron microscope stereoimage. This allows us to understand why it is that when the elongated microvilli extend or contract, the fertilization envelope also extends and contracts accordingly.     

Salinity sensitivity of early embryos of the Antarctic sea urchin, <Emphasis Type="Italic">Sterechinus neumayeri</Emphasis>

Embryos and larvae of the Antarctic sea urchin, Sterechinus neumayeri, have received considerable experimental attention assessing impacts of low temperature on development; however, salinity effects are not well documented because heretofore, the Antarctic coastal marine environment has been remarkably stenohaline. In this study, subtle decreases of 2 and 4 parts per 1,000 in standard salinity were tested to see if the developmental rate of S. neumayeri embryos would be impacted by a potential hyposmotic stress. At 30 psu, significantly fewer embryos (2 individuals out of 198 tested) reached morula stage by 36 h post-fertilization in comparison embryos in control treatments at 34 psu. Antarctic sea urchins are an important component of marine environments due to their grazing activities. Reductions in larval recruitment success due to the influx of freshwater from melting ice shelves resulting from global climate change could have far-reaching impacts on benthic ecosystem structure in Antarctica.     

Distribution and redistribution of pigment granules in the development of sea urchin embryos

Summary Pigment granules (PGs) are embeded in the cortex of embryos of the Japanese sea urchins,Hemicentrotus pulcherrimus and Anthocidaris crassispina. PGs in the cortex actively retreated from the vegetal pole area at the 4-cell stage and then a notable PG-distribution gradient formed along the egg axis (the polar redistribution of PGs). The polar redistribution of PGs in the cortex occurred at the same time after fertilization even in solutions of microtubule disrupting reagents such as Colcemid, vinblastine sulfate or griseofulvin. Consequently, the polar redistribution of PGs was not associated with the microtubules. However, the polar redistribution of PGs was interrupted in seawater containing cytochalasin B (CB), dithiothreitol (DTT) or tetracaine, and the distribution pattern of PGs in the cortex was definitely disturbed. Moreover, CB, DTT and tetracaine altered the division pattern of vegetal blastomeres at the 4th cleavage which is normally unequal so that all the blastomeres divided equally. Microtubule disrupting reagents did not have such an effect on the cleavage pattern. Thus the cortical movement along the egg axis reflected by the polar redistribution of PGs seems to correlate with the micromere formation.     

Pulse treatment of sea urchin embryos with A23187 blocks their hatching out

Summary Pulse treatment of sea urchin embryos with 3 µM A23187 for 2 h at 20° C, starting from 3 to 6 h of development, prevented the embryos from hatching. Many embryos thus treated with A23187 produced mesenchyme cells and underwent gastrulation while still enclosed within the fertilization membrane. The pulse treatment in this pre-hatching period exerts markedly stronger inhibitory effects on hatching than on other events in early development. Treatment beginning at times earlier than 2 h and later than 8 h of development caused only a slight delay of hatching. The activity of hatching enzyme, known to increase between 6 and 8 h after fertilization, was quite low, if present at all, in embryos in which hatching was blocked by A23187. Hatching enzyme synthesis is probably blocked by the preceding pulse treatment. However, overall protein synthesis, estimated with methionine S 35 incorporation, was somewhat augmented in embryos by the pulse treatment. The blockage of hatching and the augmentation of overall protein synthesis by A23187 were appreciably reversed by procaine, tetracaine, ruthenium red or verapamil. Probably, an artificial Ca²⁺ signal induced by A23187 activates protein synthesis but blocks the induction of hatching enzyme synthesis.     

Phospholipid binding properties and functional characterization of a sea urchin phospholipase Cdelta in urchin and mouse eggs

We recently identified a novel phospholipase Cdelta isoform, PLC-deltasu, in sea urchin gametes, whose precise functional role during fertilization and early embryogenesis remains unknown. Here, we characterized the binding of the PLC-deltasu PH domain to different phosphatidylinositol (PI) phospholipids and studied changes in its localization during fertilization. The PLC-deltasu PH domain bound most strongly to PI(3,4)P(2) and PI(3,5)P(2) phospholipids, in contrast to the PLCdelta1 PH domain which bound predominantly to PI(4,5)P(2). A green fluorescent protein tagged PLC-deltasu PH domain localized to the plasma membrane and its localization increased at fertilization and following addition of a Ca(2+) ionophore. However, recombinant PLC-deltasu failed to cause Ca(2+) signals like those seen at fertilization, in mouse and sea urchin eggs. Our findings suggest that PLC-deltasu is unlikely to be directly involved in the process of egg activation but may play a role in mediating extracellular signals transmitted via the PI 3'-kinase pathway.     

Direct measurement of histone peptide elongation rate in cleaving sea urchin embryos

Regulation of protein synthesis can be exercised at a number of levels. One of the more experimentally difficult levels to approach has been the measurement of peptide elongation rate. This paper presents a new application of the cyanogen bromide (CNBr) cleavage of proteins in a direct measurement of histone peptide elongation rate in cleaving sea urchin embryos (Strongylocentrotus purpuratus). The data indicate an elongation rate (at 15°C) for histones H2B and H1α of 0.69 and 0.80 codons per s, respectively. These values fall within the range of previously published values of average peptide elongation rate for total protein in these cells. This method should be generally applicable to many systems for which the measurement of peptide elongation rate may provide a key to the understanding of the regulation of protein synthesis.