Fertilized Sea Urchin Research Articles

둥근성게 (Strongylocentrotus nudus)의 수정과 초기 배발생에 미치는 빈산소의 영향

용존산소란 수생생물의 생장을 조절하는 가장 중요한 요소 중의 하나이다. 일반적으로 빈산소란 2.8 mg $O_2/L$ (91.4 mM과 동량) 이하의 용존산소로 정의된다. 따라서 빈산소 해역은 해양생태계에서 심각한 문제를 야기할 수 있다. 본 연구에서는 성게에 대한 빈산소의 배아독성 (수정 및 배발생률) 영향을 연구하기 위하여 둥근성게 (Strongylocentrotus nudus)를 용존산소 7.6 mg $O_2/L$ (normoxia)과 1.8 mg $O_2/L$ (hypoxia)에서 2일, $15^{\circ}C$ 및 33 ‰에 노출하였다. 또한 빈산소에 노출된 성게에서의 스트레스 관련유전자인 HSP70과 항산화 관련 유전자인 glutathione reductase의 발현을 immunoblotting assay와 RT-PCR을 통하여 확인하였다. 연구결과 배발생률이 현저하게 감소하였다. 분자분석 결과 빈산소에 노출된 성게가 정상상태의 성게에 비해 HSP70 단백질은 약 5.5배 glutathione reductase는 약 2.79배 증가하였다. 본 연구의 결과는 빈산소가 성게의 비정상적 배발생을 유도하며, 스트레스와 항산화 반응을 유발할 수 있음을 추측케 한다. 【Dissolved oxygen is one of the most important factors controlling growth in aquatic organisms. Hypoxia is generally defined as dissolved oxygen less than 2.8 mg $O_2/L$ (equivalent to 2 mL $O_2/L$ or 91.4 mM). Therefore, hypoxia zone can cause a serious problem in marine ecosystem. In this study, to investigate embryotoxic (fertilization and embryo development rates) effects of hypoxia on sea urchin Strongylocentrotus nudus were exposed to dissolved oxygen levels of 7.6 mg $O_2/L$ (normoxia) and 1.8 mg $O_2/L$ (hypoxia) for 2 days at $15^{\circ}C$ and 33 ‰. Also, Expression levels of stress related gene (HSP70) and antioxidant related gene (glutathione reductase) in the sea urchins exposed to hypoxia were confirmed by Immunoblotting and RT-PCR analysis. In results, we showed that developmental rates were dramatically reduced in hypoxia condition. Molecular analysis demonstrated that higher HSP70 (5.5 fold) and glutathione reductase gene (2.79 fold) were present in the sea urchin exposed to hypoxia. Our results suggested that hypoxia can cause the abnormal development and elicits a stress and antioxidant response on sea urchin.】

Fertilization in echinoderms

For more than 150years, echinoderm eggs have served as overly favored experimental model systems in which to study fertilization. Sea urchin and starfish belong to the same phylum and thus share many similarities in their fertilization patterns. However, several subtle but fundamental differences do exist in the fertilization of sea urchin and starfish, reflecting their phylogenetic bifurcation approximately 500 million years ago. In this article we review some of the seminal and recent findings that feature similarities and differences in sea urchin and starfish at fertilization.

The quest for the sea urchin egg receptor for sperm

This review discusses identification, isolation and characterization of proteins mediating species-selective sperm-to-egg adhesion during sea urchin fertilization. Bindin is the only sea urchin sperm protein known to mediate species-selective sperm attachment to eggs. Two completely different egg surface proteins, 350-kDa and EBR1, have affinity for bindin and each one meets all the criteria to be a species-selective sperm receptor. Experiments suggest that sperm bindin recognizes both the sulfated O-linked oligosaccharides on the egg 350-kDa glycoprotein, and also the repeated protein sequence modules of EBR1.

Histamine is a modulator of metamorphic competence in Strongylocentrotus purpuratus (Echinodermata: Echinoidea).

BackgroundA metamorphic life-history is present in the majority of animal phyla. This developmental mode is particularly prominent among marine invertebrates with a bentho-planktonic life cycle, where a pelagic larval form transforms into a benthic adult. Metamorphic competence (the stage at which a larva is capable to undergo the metamorphic transformation and settlement) is an important adaptation both ecologically and physiologically. The competence period maintains the larval state until suitable settlement sites are encountered, at which point the larvae settle in response to settlement cues. The mechanistic basis for metamorphosis (the morphogenetic transition from a larva to a juvenile including settlement), i.e. the molecular and cellular processes underlying metamorphosis in marine invertebrate species, is poorly understood. Histamine (HA), a neurotransmitter used for various physiological and developmental functions among animals, has a critical role in sea urchin fertilization and in the induction of metamorphosis. Here we test the premise that HA functions as a developmental modulator of metamorphic competence in the sea urchin Strongylocentrotus purpuratus.ResultsOur results provide strong evidence that HA leads to the acquisition of metamorphic competence in S. purpuratus larvae. Pharmacological analysis of several HA receptor antagonists and an inhibitor of HA synthesis indicates a function of HA in metamorphic competence as well as programmed cell death (PCD) during arm retraction. Furthermore we identified an extensive network of histaminergic neurons in pre-metamorphic and metamorphically competent larvae. Analysis of this network throughout larval development indicates that the maturation of specific neuronal clusters correlates with the acquisition of metamorphic competence. Moreover, histamine receptor antagonist treatment leads to the induction of caspase mediated apoptosis in competent larvae.ConclusionsWe conclude that HA is a modulator of metamorphic competence in S. purpuratus development and hypothesize that HA may have played an important role in the evolution of settlement strategies in echinoids. Our findings provide novel insights into the evolution of HA signalling and its function in one of the most important and widespread life history transitions in the animal kingdom - metamorphosis.

Dephosphorylation of eIF2α is essential for protein synthesis increase and cell cycle progression after sea urchin fertilization

The eukaryotic Initiation Factor 2 (eIF2) is a key regulator of protein synthesis in eukaryotic cells, implicated in the initiation step of translation. Fertilization of the sea urchin eggs triggers a rapid increase in protein synthesis activity, which is necessary for the progress into embryonic cell cycles. Here we demonstrate that fertilization triggers eIF2α dephosphorylation, concomitant with an increase in protein synthesis and that induction of the eIF2α phosphorylation is intimately linked with an inhibition of protein synthesis and cell cycle arrest. Using a phospho-mimetic protein microinjected into sea urchin eggs, we showed that dephosphorylation of eIF2α is necessary for protein synthesis activity and cell division progression following fertilization. Our results demonstrate that regulation of eIF2α plays an important role in the protein synthesis rise that occurs during early development following fertilization.

Toxicity of sediment pore water in Puget Sound (Washington, USA): a review of spatial status and temporal trends

Data from toxicity tests of the pore water extracted from Puget Sound sediments were compiled from surveys conducted from 1997 to 2009. Tests were performed on 664 samples collected throughout all of the eight monitoring regions in the Sound, an area encompassing 2,294.1 km(2). Tests were performed with the gametes of the Pacific purple sea urchin, Strongylocentrotus purpuratus, to measure percent fertilization success as an indicator of relative sediment quality. Data were evaluated to determine the incidence, degree of response, geographic patterns, spatial extent, and temporal changes in toxicity. This is the first survey of this kind and magnitude in Puget Sound. In the initial round of surveys of the eight regions, 40 of 381 samples were toxic for an incidence of 10.5 %. Stations classified as toxic represented an estimated total of 107.1 km(2), equivalent to 4.7 % of the total area. Percent sea urchin fertilization ranged from >100 % of the nontoxic, negative controls to 0 %. Toxicity was most prevalent and pervasive in the industrialized harbors and lowest in the deep basins. Conditions were intermediate in deep-water passages, urban bays, and rural bays. A second round of testing in four regions and three selected urban bays was completed 5-10 years following the first round. The incidence and spatial extent of toxicity decreased in two of the regions and two of the bays and increased in the other two regions and the third bay; however, only the latter change was statistically significant. Both the incidence and spatial extent of toxicity were lower in the Sound than in most other US estuaries and marine bays.

RNA Deep Sequencing Reveals Differential MicroRNA Expression during Development of Sea Urchin and Sea Star

microRNAs (miRNAs) are small (20–23 nt), non-coding single stranded RNA molecules that act as post-transcriptional regulators of mRNA gene expression. They have been implicated in regulation of developmental processes in diverse organisms. The echinoderms, Strongylocentrotus purpuratus (sea urchin) and Patiria miniata (sea star) are excellent model organisms for studying development with well-characterized transcriptional networks. However, to date, nothing is known about the role of miRNAs during development in these organisms, except that the genes that are involved in the miRNA biogenesis pathway are expressed during their developmental stages. In this paper, we used Illumina Genome Analyzer (Illumina, Inc.) to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of sea urchin and sea star (total of 22,670,000 reads). Analysis of these data revealed the miRNA populations in these two species. We found that 47 and 38 known miRNAs are expressed in sea urchin and sea star, respectively, during early development (32 in common). We also found 13 potentially novel miRNAs in the sea urchin embryonic library. miRNA expression is generally conserved between the two species during development, but 7 miRNAs are highly expressed in only one species. We expect that our two datasets will be a valuable resource for everyone working in the field of developmental biology and the regulatory networks that affect it. The computational pipeline to analyze Illumina reads is available at http://www.benoslab.pitt.edu/services.html.

Science and Cinema

This issue ofScience in Contextis dedicated to the question of whether there was a “cinematographic turn” in the sciences around the beginning of the twentieth century. In 1895, the Lumière brothers presented their projection apparatus to the Parisian public for the first time. In 1897, the Scottish medical doctor John McIntyre filmed the movement of a frog's leg; in Vienna, in 1898, Ludwig Braun made film recordings of the contractions of a living dog's heart (cf. Cartwright 1992); in 1904, Lucien Bull filmed in slow motion a bullet entering a soap bubble. In 1907 and 1908, respectively, Max Seddig and Victor Henri recorded Brownian motion with the help of a cinematograph (Curtis 2005). In 1909, the Swiss Julius Ries was one of the first to film fertilization and cell division in sea urchins (Ries 1909). In that same year in Paris, Louise Chevroton and Frédéric Vlès used a film camera to observe cell division in the same object (Chevroton and Vlès 1909). As early as 1898, the Parisian surgeon Eugène-Louis Doyen began filming several of his operations, among them the spectacular separation of the Siamese twins Doodica and Radica (Bonah and Laukötter 2009). And in England, the scientist and zoologist Francis Martin Duncan produced an array of popular-scientific films for Charles Urban: “The unseen world: A series of microscopic studies” was presented to the public in the Alhambra Theatre in London for the first time in 1903 (see Gaycken in this issue).

Ubiquitin-Proteasome System Involved in Fertilization of Ascidians and Sea Urchins.

Ubiquitin-Proteasome System Involved in Fertilization of Ascidians and Sea Urchins.

Identification of testis‐specific ubiquitin‐conjugating enzyme in the ascidian Ciona intestinalis

The ubiquitin-proteasome system is known to play a key role in fertilization in ascidians, sea urchins, and mammals. To obtain insights into the ubiquitin-conjugating enzymes (Ube2) involved in reproductive systems, we systematically explored Ube2 enzymes expressed in the testis of the ascidian Ciona intestinalis. Here, we report cDNA cloning and characterization of a novel type of Ube2r (Ci0100152677) that is capable of making a thiolester bond with ubiquitin. Northern analysis, whole-mount in situ hybridization and immunocytochemistry indicate that this enzyme is exclusively expressed in the testis, mainly in the germ cells during the late stage of spermatogenesis, and is localized in the sperm head and tail, suggesting possible participation in fertilization or spermatogenesis/spermiogenesis.

Sea urchin fertilization in a warm, acidified and high pCO 2 ocean across a range of sperm densities

Marine invertebrate gametes are being spawned into an ocean simultaneously warming, acidifying and increasing in pCO 2. Decreased pH/increased pCO 2 narcotizes sperm indicating that acidification may impair fertilization, exacerbating problems of sperm limitation, with dire implications for marine life. In contrast, increased temperature may have a stimulatory effect, enhancing fertilization. We investigated effects of ocean change on sea urchin fertilization across a range of sperm densities. We address two predictions: (1) low pH/increased pCO 2 reduces fertilization at low sperm density and (2) increased temperature enhances fertilization, buffering negative effects of acidification and increased pCO 2. Neither prediction was supported. Fertilization was only affected by sperm density. Increased acidification and pCO 2 did not reduce fertilization even at low sperm density and increased temperature did not enhance fertilization. It is important to identify where vulnerabilities lie across life histories and our results indicate that sea urchin fertilization is robust to climate change stressors. However, developmental stages may be vulnerable to ocean change.

Peroxidasin Is Secreted and Incorporated into the Extracellular Matrix of Myofibroblasts and Fibrotic Kidney

Mammalian peroxidases are heme-containing enzymes that serve diverse biological roles, such as host defense and hormone biosynthesis. A mammalian homolog of Drosophila peroxidasin belongs to the peroxidase family; however, its function is currently unknown. In this study, we show that peroxidasin is present in the endoplasmic reticulum of human primary pulmonary and dermal fibroblasts, and the expression of this protein is increased during transforming growth factor-beta1-induced myofibroblast differentiation. Myofibroblasts secrete peroxidasin into the extracellular space where it becomes organized into a fibril-like network and colocalizes with fibronectin, thus helping to form the extracellular matrix. We also demonstrate that peroxidasin expression is increased in a murine model of kidney fibrosis and that peroxidasin localizes to the peritubular space in fibrotic kidneys. In addition, we show that this novel pathway of extracellular matrix formation is unlikely mediated by the peroxidase activity of the protein. Our data indicate that peroxidasin secretion represents a previously unknown pathway in extracellular matrix formation with a potentially important role in the physiological and pathological fibrogenic response.

A Unique 2-Sulfated β-Galactan from the Egg Jelly of the Sea Urchin Glyptocidaris crenularis: Conformation Flexibility Versus Induction of the Sperm Acrosome Reaction

Sulfated polysaccharides from the egg jelly of sea urchins act as species-specific inducers of the sperm acrosome reaction, which is a rare molecular mechanism of carbohydrate-induced signal-transduction event in animal cells. The sea urchin polysaccharides differ in monosaccharide composition (l-fucose or l-galactose), glycosylation, and sulfation sites, but they are always in the alpha-anomeric configuration. Herein, structural analysis of the polysaccharide from the sea urchin Glyptocidaris crenularis surprisingly revealed a unique sulfated beta-d-galactan composed by (3-beta-d-Galp-2(OSO(3))-1-->3-beta-d-Galp-1)(n) repeating units. Subsequently, we used the G. crenularis galactan to compare different 2-sulfated polysaccharides as inducers of the acrosome reaction using homologous and heterologous sperm. We also tested the effect of chemically over-sulfated galactans. Intriguingly, the anomeric configuration of the glycosidic linkage rather than the monosaccharide composition (galactose or fucose) is the preferential structural requirement for the effect of these polysaccharides on sea urchin fertilization. Nuclear magnetic resonance and molecular dynamics indicate that sulfated alpha-galactan or alpha-fucan have less dynamic structural behavior, exhibiting fewer conformational populations, with an almost exclusive conformational state with glycosidic dihedral angles Phi/Psi = -102 degrees /131 degrees . The preponderant conformer observed in the sulfated alpha-galactan or alpha-fucan is not observed among populations in the beta-form despite its more flexible structure in solution. Possibly, a proper spatial arrangement is required for interaction of the sea urchin-sulfated polysaccharides with the specific sperm receptor.

Review: An overview about the structure–function relationship of marine sulfated homopolysaccharides with regular chemical structures

Efforts in both structural and biological studies of sulfated polysaccharides from marine organisms have increased significantly over the last 10 years. Marine invertebrates have been demonstrated to be a source of glycans with particularly well-defined chemical structures, although ordered structural patterns can also be found occasionally in algal sources such as red seaweeds. Clear and regular structural features are essential for a good understanding of the biological activities of these marine homopolysaccharides of which sulfated fucans and sulfated galactans are the most studied. Herein, the main structural features (sugar type, sulfation and glycosylation sites, and orientational binding preferences) of both sulfated fucans and galactans are individually reviewed with regard to their specific contributions to two frequently described biological functions: the acrosome reaction (a physiological event of sea-urchin fertilization), and the anticoagulant and antithrombotic activities (an alternative and highly desirable pharmacological application). (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 601-609, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.

SpH‐protease exists in Caco‐2 cells

We have described a 60 kDa nuclear cysteine protease that degrades sperm histones during male chromatin remodeling after fertilization in sea urchins (SpH protease). This enzyme was shown to be localized in the mitotic furrow and to be involved in cell cycle control in sea urchin embryos. The N terminal sequence and the gene encoding this protease have homology with human cathepsin L family; therefore we investigated with antibodies raised against the sea urchins protease if a similar protease is present in Caco‐2 cells. Among Caco 2 chromosomal proteins obtained from confluent cell cultures, we found by Western blots, two bands migrating at 60 and 100 kDa in SDS/PAGE. We have also used these anti SpH protease antibodies to immunolocalize this protease in the intracellular context. This protease in mitotic cells localize on the mitotic furrow and remains in the nucleus in binuclear cells. On the other hand, when inhibitors of cysteine‐proteasas were used in different phases of the cell cycle we observed a disorganization of the mitotic spindle and an alteration in the localization of the protease during the S phase affecting DNA replication. Our results demonstrate that in Caco 2 cells exist a variant of cathepsin L protease that exhibits a similar localization to that was described for embryos of sea urchin, indicating a putative rol in cell proliferation. FONDECYT 11070067.

Temperature, but not pH, compromises sea urchin fertilization and early development under near-future climate change scenarios.

Global warming is causing ocean warming and acidification. The distribution of Heliocidaris erythrogramma coincides with the eastern Australia climate change hot spot, where disproportionate warming makes marine biota particularly vulnerable to climate change. In keeping with near-future climate change scenarios, we determined the interactive effects of warming and acidification on fertilization and development of this echinoid. Experimental treatments (20-26 degrees C, pH 7.6-8.2) were tested in all combinations for the 'business-as-usual' scenario, with 20 degrees C/pH 8.2 being ambient. Percentage of fertilization was high (>89%) across all treatments. There was no difference in percentage of normal development in any pH treatment. In elevated temperature conditions, +4 degrees C reduced cleavage by 40 per cent and +6 degrees C by a further 20 per cent. Normal gastrulation fell below 4 per cent at +6 degrees C. At 26 degrees C, development was impaired. As the first study of interactive effects of temperature and pH on sea urchin development, we confirm the thermotolerance and pH resilience of fertilization and embryogenesis within predicted climate change scenarios, with negative effects at upper limits of ocean warming. Our findings place single stressor studies in context and emphasize the need for experiments that address ocean warming and acidification concurrently. Although ocean acidification research has focused on impaired calcification, embryos may not reach the skeletogenic stage in a warm ocean.

SEDIMENT CONTAMINANT AND TOXICITY IN KELABAT BAY, BANGKA BELITUNG PROVINCE

Sediment of 16 sites in the Kelabat Bay were monitored for metal contamination and toxicity in 2006. Two patterns of metal contaminants distribution in the sediment were observed. Higher concentrations of lead and copper were found in March than those in July, and so was in the inner bay than those observed in the outer bay, while different pattern was observed for cadmium concentration. The highest lead concentrations were observed at site 7 (inner bay) both in March and July. Although elevated levels of cadmium, lead and copper were observed in sediments, they were still in the tolerable level for marine life. Sediment of the bay was also evaluated for its toxicity using marine phytoplankton, Chaetoceros gracilis and sea urchin, Tripneustes gratila. Results of the tests reveal that no toxic effects of sediment to the growth of phytoplankton were observed, but to sea urchin fertilization of the sediment may have toxic effect.

Germ Cell Research: A Personal Perspective

My interest in germ cells began when I first witnessed sea urchin fertilization and embryo development during a laboratory class at Hokkaido University, Sapporo, Japan, almost 60 yr ago. Weismann's concept of germ cells that I learned during my undergraduate years became the driving force of my entire research career. During the early years, my associates and I used mainly the golden hamster and the guinea pig as model animals because their spermatozoa had large acrosomes and we could readily follow changes in the acrosomes without killing or staining spermatozoa. We later used the mouse as our model organism because we wanted to produce live offspring with known genetic backgrounds. A summary of the findings we made during those years includes the following: (1) first in vitro sperm capacitation; (2) discovery of sperm hyperactivation; (3) demonstration of the importance of Ca(2+) in sperm acrosome reaction, hyperactivation, sperm-egg fusion, and egg activation; (4) development of the sperm's fusion competence during the acrosome reaction; (5) characterization of sperm-oviduct relationships before and during fertilization; (6) use of zona-free hamster eggs to examine fertilizing ability and chromosomes of human spermatozoa; (7) development and use of intracytoplasmic sperm injection; (8) use of prespermatozoal cells for the production of offspring; (9) sperm preservation by freeze-drying and freezing whole-animal bodies; and (10) mouse cloning by somatic cell nuclear transfer. My current interests and my visions for the future include the following: (1) mass production of mature eggs and spermatozoa in vitro, (2) permanent sperm preservation at ambient temperature, (3) development of safer and more efficient assisted fertilization (reproduction) technologies, (4) development of safe and efficient methods of cloning, (5) production of artificial organs such as an artificial uterus, (6) development of safe and effective male contraceptives, and (7) prevention of cancer through germ cell research.

Lipid levels in sperm, eggs, and during fertilization in Xenopus laevis

Critical developmental periods, such as fertilization, involve metabolic activation, membrane fusion events such as sperm-egg or plasma membrane-cortical granule merger, and production and hydrolysis of phospholipids. However, there has been no large-scale quantification of phospholipid changes during fertilization. Using an enzymatic assay, traditional FA analysis by TLC and gas chromatography, along with a new method of phospholipid measurement involving HPLC separation and evaporative light-scattering detection, we report lipid levels in eggs, sperm, and during fertilization in Xenopus laevis. Sperm were found to contain different amounts of phospholipids as compared with eggs. During fertilization, total phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, and phosphatidylserine decreased, and ceramide increased, whereas there was no change in phosphatidylcholine, cardiolipin, or phosphatidylethanolamine. FA analysis of phospholipids found numerous changes during fertilization. Because there is an increase in sn-1,2-diacylglycerol at fertilization, the FAs associated with this increase and the source of the increase in this neutral lipid were examined. Finally, activation of phospholipase C, phospholipase D, phospholipase A2, autotoxin, and sphingomyelinase at fertilization is discussed.

Ca2+ Signaling Occurs via Second Messenger Release from Intraorganelle Synthesis Sites

SummaryCyclic ADP-ribose is an important Ca2+-mobilizing cytosolic messenger synthesized from β-NAD+ by ADP-ribosyl cyclases (ARCs). However, the focus upon ectocellular mammalian ARCs (CD38 and CD157) has led to confusion as to how extracellular enzymes generate intracellular messengers in response to stimuli. We have cloned and characterized three ARCs in the sea urchin egg and found that endogenous ARCβ and ARCγ are intracellular and located within the lumen of acidic, exocytotic vesicles, where they are optimally active. Intraorganelle ARCs are shielded from cytosolic substrate and targets by the organelle membrane, but this barrier is circumvented by nucleotide transport. We show that a β-NAD+ transporter provides ARC substrate that is converted luminally to cADPR, which, in turn, is shuttled out to the cytosol via a separate cADPR transporter. Moreover, nucleotide transport is integral to ARC activity physiologically because three transport inhibitors all inhibited the fertilization-induced Ca2+ wave that is dependent upon cADPR. This represents a novel signaling mechanism whereby an extracellular stimulus increases the concentration of a second messenger by promoting messenger transport from intraorganelle synthesis sites to the cytosol.