Experimentally observed soft‐tissue preservation near a marine brine seep

A sea urchin placed on the sea floor near an active brine seep was recovered after 13 years with detailed soft‐tissue preservation. Growth of an amorphous calcium carbonate solid with small amounts of the mineral bassanite occurred on the spines and test. The solid also exhibits striations at both the macro‐ and microscopic scales that preserve the muscle texture of the sea urchin. Such soft‐tissue replacement and mineralization could lead to exquisite fossilization. Soft‐tissue mineralization has been previously replicated in controlled laboratory conditions; however, this is the first report of the lithologic replication of soft tissues in an open marine experiment. Examples of extraordinary fossil preservation, or Lagersätten, give a distinct snapshot of the past and have led to a greater understanding of the history of life. Soft‐tissue lithification occurs in special circumstances in which local chemical conditions (often mediated by decay or bacterial factors) promote early diagenetic mineralization, the first steps of which are observed in this instance. The preservation of articulated skeletons, especially within echinoderms, is normally attributed to rapid burial, but that may not be necessary given that this urchin was at or very near the sediment–water interface for 13 years.     

Immunological evidence for the localization of sialoglycosphingolipids at the cell surface of sea urchin spermatozoa.

The localization of sialoglycosphingolipids in the plasma membrane of sea urchin spermatozoa was studied by employing immunological methods including immunolysis of liposomal model membranes. The antibodies produced against these complex lipids were found to agglutinate various sea urchin spermatozoa differently. Both species differences and species similarities in the agglutination were found in spermatozoa of the echinoderm, the sea urchin and the starfish. The agglutination of the sea urchin spermatozoa was inhibited specifically by ceratain carbohydrates. Only a limited number of molecular species of sialoglycosphingolipid were localized at the surface of the plasma membrane of sea urchin spermatozoa cells. Moreover, topographical differences were found in the localization of the sialoglycosphingolipids at the cell surface of spermatozoa.     

Studies of the mechanism of the electrical polyspermy block using voltage clamp during cross-species fertilization

Prevention of polyspermic fertilization in sea urchins (Jaffe, 1976, Nature (Lond.). 261:68-71) and the worm Urechis (Gould-Somero, Jaffe, and Holland, 1979, J. Cell Biol. 82:426-440) involves an electrically mediated fast block. The fertilizing sperm causes a positive shift in the egg's membrane potential; this fertilization potential prevents additional sperm entries. Since in Urechis the egg membrane potential required to prevent fertilization is more positive than in the sea urchin, we tested whether in a cross-species fertilization the blocking voltage is determined by the species of the egg or by the species of the sperm. With some sea urchin (Strongylocentrotus purpuratus) females, greater than or equal to 90% of the eggs were fertilized by Urechis sperm; a fertilization potential occurred, the fertilization envelope elevated, and sometimes decondensing Urechis sperm nuclei were found in the egg cytoplasm. After insemination of sea urchin eggs with Urechis sperm during voltage clamp at +50 mV, fertilization (fertilization envelope elevation) occurred in only nine of twenty trials, whereas, at +20 mV, fertilization occurred in ten of ten trials. With the same concentration of sea urchin sperm, fertilization of sea urchin eggs occurred, in only two of ten trials at +20 mV. These results indicate that the blocking voltage for fertilization in these crosses is determined by the sperm species, consistent with the hypothesis that the fertilization potential may block the translocation within the egg membrane of a positively charged component of the sperm.     

Elongated Microvilli on Vegetal Pole Cells in Sea Urchin Embryos

The ultrastructure of cells in the vegetal pole region of sea urchin embryos during early development to the mesenchyme blastula stage was examined by scanning electron microscopy. Vegetal pole cells in the ectoderm with longer microvilli than those of neighboring cells were first detectable at the early blastula stage just before hatching. These cells with elongated microvilli remained in the central region of the vegetal plate when most vegetal plate cells ingressed into the blastocoel to form primary mesenchyme. When first detectable in the sea urchin, Anthocidaris crassispina , four vegetal pole cells had elongated microvilli, but at the time of primary mesenchyme cell ingression, the number of cells with elongated microvilli had increased to eight, apparently by cell division. These vegetal pole cells were wedge-shaped with a broad surface adhering to the hyaline layer at the time of primary mesenchyme cell ingression. SEM observation of the outer surface of embryos showed that the microvilli extended into the hyaline layer. The reinforced attachment of vegetal pole cells to the hyaline layer through their elongated microvilli may explain why these cells could remain at the vegetal pole when the surrounding cells ingressed into the blastocoel as primary mesenchyme cells.     

The S. purpuratus genome: a comparative perspective

The predicted gene models derived from the sea urchin genome were compared to the gene catalogs derived from other completed genomes. The models were categorized by their best match to conserved protein domains. Identification of potential orthologs and assignment of sea urchin gene models to groups of homologous genes was accomplished by BLAST alignment and through the use of a clustering algorithm. For the first time, an overview of the sea urchin genetic toolkit emerges and by extension a more precise view of the features shared among the gene catalogs that characterize the super-clades of animals: metazoans, bilaterians, chordate and non-chordate deuterostomes, ecdysozoan and lophotrochozoan protostomes. About one third of the 40 most prevalent domains in the sea urchin gene models are not as abundant in the other genomes and thus constitute expansions that are specific at least to sea urchins if not to all echinoderms. A number of homologous groups of genes previously restricted to vertebrates have sea urchin representatives thus expanding the deuterostome complement. Obversely, the absence of representatives in the sea urchin confirms a number of chordate specific inventions. The specific complement of genes in the sea urchin genome results largely from minor expansions and contractions of existing families already found in the common metazoan "toolkit" of genes. However, several striking expansions shed light on how the sea urchin lives and develops.     

Cyclin E/Cdk2 is required for sperm maturation, but not DNA replication, in early sea urchin embryos

The cell cycle is driven by the activity of cyclin/cdk complexes. In somatic cells, cyclin E/cdk2 oscillates throughout the cell cycle and has been shown to promote S-phase entry and initiation of DNA replication. In contrast, cyclin E/cdk2 activity remains constant throughout the early embryonic development of the sea urchin and localizes to the sperm nucleus following fertilization. We now show that cyclin E localization to the sperm nucleus following fertilization is not unique to the sea urchin, but also occurs in the surf clam, and inhibition of cyclin E/cdk2 activity by roscovitine inhibits the morphological changes indicative of male pronuclear maturation in sea urchin zygotes. Finally, we show that inhibition of cyclin E/cdk2 activity does not block DNA replication in the early cleavage cycles of the sea urchin. We conclude that cyclin E/cdk2 activity is required for male pronuclear maturation, but not for initiation of DNA replication in early sea urchin development.     

Molecular cloning and characterization of the mRNA for cyclin from sea urchin eggs.

We have isolated a cDNA clone encoding sea urchin cyclin and determined its sequence. It contains a single open reading frame of 409 amino acids which shows homology with clam cyclins. RNA transcribed in vitro from this sequence was efficiently translated in reticulocyte lysates, yielding full-length cyclin. Injection of nanogram amounts of this synthetic mRNA into Xenopus oocytes caused them to mature more rapidly than with progesterone treatment. The sea urchin cyclin underwent two posttranslational modifications in the Xenopus oocytes during maturation. The first occurred at about the time that maturation became cycloheximide-resistant, when a small apparent increase in the molecular weight of cyclin was observed. The second modification involved destruction of the cyclin at about the time of white spot appearance, just as would have occurred at the metaphase/anaphase transition in the natural environment of a cleaving sea urchin embryo.     

Kohamaic acid A,a novel sesterterpenic acid,inhibits activities of DNA polymerases from deuterostomes

We previously found and isolated a novel natural product, designated kohamaic acid A (KA-A), which inhibited the first cleavage of fertilized sea urchin eggs. In this paper, we report that this compound could selectively inhibit the activities of DNA polymerases (pol. alpha, beta, gamma, delta and epsilon ) only from species in the deuterostome branch in the animal kingdom, like sea urchin, fish and mammals, but not from protostomes including insects (fruit fly, Drosophila melanogaster) and mollusks (octopus and oyster). Inhibition of deuterostome DNA polymerases was dose dependent. IC(50) values for DNA polymerases of mammals and fish occurred at approximately 5.8-14.9 microM and those of sea urchin at 6.1-30.3 microM. In the sea urchin DNA polymerases, the activities of the replicative DNA polymerases such as alpha, delta and epsilon were more strongly inhibited than that of the repair-related pol. beta. KA-A is an inhibitor of replicative DNA polymerases from the deuterostome species, and subsequently, the inhibition of the first cleavage of fertilized sea urchin eggs might occur as a result of the suppression of DNA replication.     

ULTRASTRUCTURE AND BIREFRINGENCE OF THE ISOLATED MITOTIC APPARATUS OF MARINE EGGS

Isolated mitotic apparatuses (MA) of clam and sea urchin eggs were investigated by polarizing and electron microscopy. Examination of fixed MA in oils of different refractive index revealed that at least 90% of the retardation of isolated MA is due to positive, form birefringence, the remaining retardation deriving from positive, intrinsic birefringence. Electron micrographs reveal the isolated MA to be composed of microtubules, ribosome-like particles, and a variety of vesicles. In the clam MA the number of vesicles and ribosome-like particles relative to the number of microtubules is much lower than in the sea urchin MA. In clam MA this allows form and intrinsic birefringence to be related directly to microtubules. The relation of birefringence to microtubules in isolated sea urchin MA is more complex since ribosome-like particles adhere to microtubules, are oriented by them, and are likely to contribute to the form birefringence of the isolated MA. However, comparison of values of retardation for clam and sea urchin MA, indicates that the major part of the birefringence in sea urchin MA is also due to microtubules. The interpretation of the structures giving rise to birefringence in the MA of the living cells is likely to be even more complex since masking substances, compression, or tension on the living MA may alter the magnitude or sign of the birefringence.     

Behavior and differentiation process of pigment cells in a tropical sea urchin Echinometra mathaei

The behavior and differentiation processes of pigment cells were studied in embryos of a tropical sea urchin Echinometra mathaei, whose egg volume was one half of those of well-known sea urchin species. Owing to earlier accumulation of pigments, pigment cells could be detected in the vegetal plate even before the onset of gastrulation, distributed dorsally in a hemi-circle near the center of the vegetal plate. Although some pigment cells left the archenteron during gastrulation, most of them remained at the archenteron tip. At the end of gastrulation, pigment cells left the archenteron and migrated into the blastocoele. Unlike pigment cells in typical sea urchins, however, they did not enter the ectoderm, and stayed in the blastocoele even at the pluteus stage. It is of interest that the majority of pigment cells were distributed in the vicinity of the larval skeleton. Aphidicolin treatment revealed that eight blastomeres were specific to pigment cell lineage after the eighth cleavage, one cell cycle earlier than that in well-known sea urchins. The pigment founder cells divided twice, and the number of pigment cells was around 32 at the pluteus stage. It was also found that the differentiation of pigment cells was blocked with Ni2+, whereas the treatment was effective only during the first division cycle of the founder cells.     

Male chromosomes of sea urchin hybrid andromerogones created with cryopreserved sperm

We developed a method for preparing male chromosomes from sea urchin hybrid andromerogones created with cryopreserved sperm. We obtained hybrid andromerogones by heterospermic insemination of Hemicentrotus pulcherrimus non-nucleate egg fragments produced by centrifuging unfertilized eggs in a stepwise saccharose density gradient. The hybrid andromerogones showed cleavage rates of 1%-93%, cleaved successively into two- and four- blastomeres and developed to early blastulae. The morulae or early blastulae were treated with colchicine (0.1-1.0 mg/ml), dissociated into single blastomeres by pippeting, swollen with 7%-10% sodium citrate for 10 min and fixed with methanol:acetic acid (3:1). The fixed cells were dropped on slides and air-dried. The andromerogones for 5 sperm species showed a half of their respective diploid chromosome numbers without chromosome elimination. This method is applicable for analysis of the haploid male chromosome complement in sea urchin species for which only sperm can be obtained.     

Microtubule assembly is required for the formation of the pronuclei,nuclear lamin acquisition,and DNA synthesis during mouse,but not sea urchin,fertillization

Microtubule assembly is required for the formation of the male and female pronuclei during mouse, but not sea urchin, fertilization. In mouse oocytes, 50 μM colcemid prevents the decondensation of the maternal meiotic chromosomes and of the incorporated sperm nucleus during in vitro fertilization. Nuclear lamins do not associate with either of the parental chromatin sets although peripherin, the PI nuclear peripheral antigen, appears on both. DN A synthesis docs not occur in these fertilized, colcemid-arrested oocytes. This effect is limited to the first hours after ovulation, since colcemid added 4–6 hours later no longer prevents pronuclear development, lamin acquisition, or DNA synthesis. Neither microtubule stabilization with 10 μM taxol nor microfilament inhibition with 10 μM cytochalasin D or 2.2 μg/ml lalrunculin A prevent these pronuclear events; these drugs will inhibit the apposition of the pronuclei at the egg center. In sea urchin eggs, colcemid or griseofulvin treatment doe? not result in the same effect and the male pronucleus forms with the attendant accumulation of the nuclear lamins. The differences in the requirement for microtubule assembly during pronucleus formation may be related to the cell cycle: In mice the sperm enters a meiotic cytoplasm, whereas in sea urchin eggs it enters an interphase cytoplasm. Refertilization of mitotic sea urchin eggs was performed to test the possibility that this phenomenon is related to whether the sperm enters a meiotic/mitotic cytoplasm or one at interphase; during refertilization at first mitosis, the incorporated sperm nucleus is unable to decondense and acquire lamins. These results indicate a requirement for microtubule assembly for the progression from meiosis to first interphase during mouse fertilization and suggest that the cytoskeleton is required for changes in nuclear architecture necessary during fertilization and the cell cycle.     

Redescription of Syndesmis echinorum Fran?ois, 1886 (Turbellaria: Neorhabdocoela: Umagillidae), with comments on distinctions between Syndesmis and Syndisyrinx

Syndesmis echinorum is redescribed from specimens taken from the intestine of the sea urchin Paracentrotus lividus at the type locality, Banyuls-sur-Mer, France. This umagillid is shown to have a seminal bursa-bursal valve complex comparable to that of Syndisyrinx franciscanus. Thus, it is suggested that the genera Syndesmis and Syndisyrinx be distinguished on the basis of the form and proportions of the ejaculatory duct, male antrum, and penis stylet, rather than on the basis of the presence or absence of a seminal bursa and bursal valve. The sea urchin Sphaerechinus granularis at Banyuls harbors a worm that is, on the average, slightly more robust than S. echinorum from P. lividus; its morphology, however, agrees with that of S. echinorum. In addition to S. echinorum, P. lividus harbors at least 1 undescribed umagillid. Although 2 species found in Echinus esculentus have been referred to S. echinorum by previous workers, neither is conspecific with it.     

Long-Term Changes in the Gonad Condition of the Sea Urchin Strongylocentrotus intermediusfrom Alekseev Bight (Amurskii Bay) under Polluted Conditions

The gonad condition of the sea urchin Strongylocentrotus intermediuscollected in August 1997 at two stations in Peter the Great Bay was examined. One of the stations was located in a polluted area (Alekseev Bight, Popov Island) and the other, in a relatively clean area (the Verkhovskii Islands). The results were compared with analogous data for 1984, 1985, and 1989. In 1997, the gonad condition of sea urchins inhabiting the two areas differed significantly. The mean value of the gonad index (GI) for sea urchins from Alekseev Bight was less than half and the maturity index was about twice that of sea urchins from the Verkhovskii Islands. The GI of sea urchins from Alekseev Bight decreased by a factor of 1.5 between 1984 and 1997. Pronounced histopathological changes were found in sea urchin gonads at this station: granular and hydropic dystrophy of oocytes, resorption and a sharp decrease in the number of gametes (in about 20% of the sea urchins, hardly any gametes were found in the gonads), changes in the morphology of accessory cells (hypertrophy, atrophy, and necrosis), and accumulation of lipofuscin in the cytoplasm of accessory cells and oocytes, in the hemal sinuses and mesentery. The suppressed gonad condition of the sea urchin S. intermediusin Alekseev Bight may be a consequence of the unfavorable environmental situation that formed in the bight in the 1980s–1990s. The main negative factor is anthropogenic pollution of Amurskii Bay.     

A method of microinjection: delivering monoclonal antibody 1223 into sea urchin embryos.

In this paper, a simpler method of microinjecting sea urchin embryos without using the conventional microinjection chamber designed by Kiehart is reported. A trough was made on a surface of 0.6% agarose gel dissolved in artificial sea water. Approximately fifty hatched embryos could be loaded in the trough and, consequently, swimming embryos were trapped in the trough. Monoclonal antibody (mAB) 1223 which blocks spiculogenesis in vitro was delivered into the blastocoels of sea urchin embryos to test whether this antibody inhibits spiculogenesis in vivo and also, whether this new technique is effective for the microinjection of the sea urchin embryos. The embryos were injected with mAB1223 at the hatched blastula, early mesenchyme blastula and early gastrula stages, and 63%, 90% and 97% of the embryos did not form spicules at the late gastrula stage, respectively. Therefore, mAB1223 was shown to also block spiculogenesis in vivo. From the fact that spiculogenesis occurred at a lower rate when mAB1223 was injected at the hatched blastula stage than at later stages, it may be speculated that endogenous proteases degraded the injected antibodies. Using this technique, extracellular events in the blastocoel or the function of certain molecules expressed in blastocoel can be easily investigated in vivo.