Fertilized Sea Urchin Eggs Research Articles

Species-Specific Gamete Interaction during Sea Urchin Fertilization: Roles of the Egg Jelly and Vitelline Layer

In sea urchins, the sequence of the cellular and molecular events characterizing the fertilization process has been intensively studied. We have learned that to activate the egg, the fertilizing sperm must undergo morphological modifications (the acrosome reaction, AR) upon reaching the outer gelatinous layer enveloping the egg (egg jelly), which triggers the polymerization of F-actin on the sperm head to form the acrosomal process. The AR exposes bindin, an adhesive sperm protein essential for the species-specific interaction with the cognate receptor on the egg vitelline layer. To investigate the specific roles of the egg jelly and vitelline layer at fertilization of sea urchin eggs, Paracentrotus lividus eggs were incubated in acidic seawater, which removes the egg jelly, i.e., experimental conditions that should prevent the occurrence of the AR, and inseminated in the same medium. At variance with the prevailing view, our results have shown that these dejellied P. lividus eggs can still interact with sperm in acidic seawater, albeit with altered fertilization responses. In particular, the eggs deprived of the vitelline layer reacted with multiple sperm but with altered Ca2+ signals. The results have provided experimental evidence that the plasma membrane, and not the vitelline layer, is where the specific recognition between gametes occurs. The vitelline layer works in unfertilized eggs to prevent polyspermy.

Sodium-mediated fast electrical depolarization does not prevent polyspermic fertilization in Paracentrotus lividus eggs.

During sea urchins fertilization, the activating spermatozoon triggers a series of physiological changes that transforms the quiescent egg into a dynamic zygote. It has been suggested that several of these egg activation events, e.g. sperm-induced plasma membrane depolarization and the Ca2+-linked cortical reaction, play additional roles to prevent the entry of supernumerary spermatozoa. In particular, the abrupt shift in egg membrane potential at fertilization, which is sustained by a Na+ influx, has been considered as a fast mechanism to block polyspermy. To test the relevance of the Na+-mediated fast electrical block to polyspermy, we fertilized sea urchin eggs in artificial seawater with a low concentration of Na+; nearly all the eggs were still monospermic, as judged by the number of Hoechst 33422-stained sperm. When fertilized in normal seawater, eggs that were pre-incubated in the low Na+ medium exhibited impaired elevation of the fertilization envelope. Nevertheless, these eggs manifested entry of a single spermatozoon, suggesting that the fertilization envelope was not the primary determinant of the block to polyspermy. Furthermore, we showed that the abnormal cleavage patterns displayed by eggs pre-incubated in low Na+, which were often considered a hallmark of polyspermy, were due to the alterations in the cortical actin filaments dynamics following fertilization, and not to the formation of multipolar spindles associated with supernumerary sperm centrosomes. Hence, our results suggested that Paracentrotus lividus eggs do not utilize Na+ to rapidly prevent additional spermatozoa from entering the egg, at variance with the hypothesis of an electrical fast block to polyspermy.

Fertilization in Starfish and Sea Urchin: Roles of Actin.

Marine animals relying on "external fertilization" provide advantageous opportunities to study the mechanisms of gamete activation and fusion, as well as the subsequent embryonic development. Owing to the large number of eggs that are easily available and handled, starfish and sea urchins have been chosen as favorable animal models in this line of research for over 150years. Indeed, much of our knowledge on fertilization came from studies in the echinoderms. Fertilization involves mutual stimulation between eggs and sperm, which leads to morphological, biochemical, and physiological changes on both sides to ensure successful gamete fusion. In this chapter, we review the roles of actin in the fertilization of starfish and sea urchin eggs. As fertilization is essentially an event that takes place on the egg surface, it has been predicted that suboolemmal actin filaments would make significant contributions to sperm entry. A growing body of evidence from starfish and sea urchin eggs suggests that the prompt reorganization of the actin pools around the time of fertilization plays crucial regulatory roles not only in guiding sperm entry but also in modulating intracellular Ca2+ signaling and egg activation.

Light-stick: A problem of marine pollution in Brazil

Light-sticks are used as bait in surface long-line fishing, to capture swordfish and other large pelagic predators. When discharged in the ocean, it may reach the beaches. The traditional Brazilian community of Costa dos Coqueiros, Bahia, use light-sticks as a medicine for rheumatism, vitiligo and mycoses. It may affect the marine life when its content leak in the open ocean. This work evaluated and identified the acute and chronic toxicity of the light-stick. A high acute toxicity was observed in the mobility/mortality of Artemia sp.; in the fertilization of sea urchin eggs, and a high chronic toxicity in the development of the pluteus larvae of the same sea urchin. The main compounds that probably caused toxicity were the volatiles such as the fluorescent PAH and oxidants such as the hydrogen peroxide. Its disposal in the open ocean is a potential threat for marine life.

Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos.

The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism.

Early events of fertilization in sea urchin eggs are sensitive to actin-binding organic molecules

We previously demonstrated that many aspects of the intracellular Ca2+ increase in fertilized eggs of starfish are significantly influenced by the state of the actin cytoskeleton. In addition, the actin cytoskeleton appeared to play comprehensive roles in modulating cortical granules exocytosis and sperm entry during the early phase of fertilization. In the present communication, we have extended our work to sea urchin which is believed to have bifurcated from the common ancestor in the phylogenetic tree some 500 million years ago. To corroborate our earlier findings in starfish, we have tested how the early events of fertilization in sea urchin eggs are influenced by four different actin-binding drugs that promote either depolymerization or stabilization of actin filaments. We found that all the actin drugs commonly blocked sperm entry in high doses and significantly reduced the speed of the Ca2+ wave. At low doses, however, cytochalasin B and phalloidin increased the rate of polyspermy. Overall, certain aspects of Ca2+ signaling in these eggs were in line with the morphological changes induced by the actin drugs. That is, the time interval between the cortical flash and the first Ca2+ spot at the sperm interaction site (the latent period) was significantly prolonged in the eggs pretreated with cytochalasin B or latrunculin A, whereas the Ca2+ decay kinetics after the peak was specifically attenuated in the eggs pretreated with jasplakinolide or phalloidin. In addition, the sperm interacting with the eggs pretreated with actin drugs often generated multiple Ca2+ waves, but tended to fail to enter the egg. Thus, our results indicated that generation of massive Ca2+ waves is neither indicative of sperm entry nor sufficient for cortical granules exocytosis in the inseminated sea urchin eggs, whereas the structure and functionality of the actin cytoskeleton are the major determining factors in the two processes.

Modelization of the regulation of protein synthesis following fertilization in sea urchin shows requirement of two processes: a destabilization of eIF4E:4E-BP complex and a great stimulation of the 4E-BP-degradation mechanism, both rapamycin-sensitive.

Fertilization of sea urchin eggs involves an increase in protein synthesis associated with a decrease in the amount of the translation initiation inhibitor 4E-BP. A highly simple reaction model for the regulation of protein synthesis was built and was used to simulate the physiological changes in the total 4E-BP amount observed during time after fertilization. Our study evidenced that two changes occurring at fertilization are necessary to fit with experimental data. The first change was an 8-fold increase in the dissociation parameter (koff1) of the eIF4E:4E-BP complex. The second was an important 32.5-fold activation of the degradation mechanism of the protein 4E-BP. Additionally, the changes in both processes should occur in 5 min time interval post-fertilization. To validate the model, we checked that the kinetic of the predicted 4.2-fold increase of eIF4E:eIF4G complex concentration at fertilization matched the increase of protein synthesis experimentally observed after fertilization (6.6-fold, SD = 2.3, n = 8). The minimal model was also used to simulate changes observed after fertilization in the presence of rapamycin, a FRAP/mTOR inhibitor. The model showed that the eIF4E:4E-BP complex destabilization was impacted and surprisingly, that the mechanism of 4E-BP degradation was also strongly affected, therefore suggesting that both processes are controlled by the protein kinase FRAP/mTOR.

Multiscale Modeling of Exocytosis in the Fertilization Process

We discuss the implementation of a multiscale biophysico-chemical model able to cope with the main mechanisms underlying cumulative exocytosis in cells. The model is based on a diffusion equation in the presence of external forces that links calcium signaling and the biochemistry associated to the activity of cytoskeletal-based protein motors. This multiscale model offers an excellent quantitative spatio-temporal description of the cumulative exocytosis measured by means of fluorescence experiments. We also review pre-existing models reported in the literature on calcium waves, protein motor activation and dynamics, and intracellular directed transport of vesicles. As an example of the proposed model, we analyze the formation of the shield against polyspermy in the early events of fertilization in sea urchin eggs.

Multiscale Model of the Propagation of the Fertilization Wave in Sea Urchin Eggs

We present a multiscale model describing blocking polyspermy during the fertilization in sea urchin eggs [1,2]. Our goal is to describe the processes controlling the formation of the vitelline envelope as a consequence of hyaline exocytosis. Our model combines two different levels of description: a) A reaction-diffusion scheme based on the CICR model [3], accounting for the initial propagation of the calcium wave, its duration and spatial distribution in the cell. b) The activation and dynamics of kinesin motors transporting vesicle clusters that contain hyaline, triggered due to calcium signaling. Our model incorporates a recent biochemical model [4] describing the kinetics of kinesin processivity in order to reproduce observed translational velocities, their duration and dependence on local ATP concentration. In addition, this dynamics is incorporated into a diffusion equation in the presence of external forces that accounts for the motions of vesicle clusters along the citoskeletal filaments of the egg [5]. Our model reproduces very well experimental data associated to the propagation velocities of the calcium wave, the kinesins and the vesicle clusters, as well as their duration and the kinetics of fluorescence used to indirectly quantify the cumulative exocytosis of hyaline at the plasmatic membrane.[1] D. J. Bonder, E. M. Fishkind, Actin-Membrane citoskeletal dynamics in early sea urchin development, in Citoskeletal mechanism during animal development, Ed. G. Capco (Academic Press Inc. 1995).[2] ALedesma-Duran, I. Santamaria-Holek, Multiscale model of the propagation of the fertilization wave in sea urchin eggs, in preparation.[3] J. P. Keener, J. Sneyd, Interdisciplinary mathematics 8, Springer, Berlin (1998).[4] N.J. Lopez Alamilla, I. Santamaria-Holek, Biophys. Chem. 167, 16-25 (2012).[5] F. F. de Miguel, I. Santamaria-Holek et al, Biophysics of active vesicle transport, accepted in PLoS ONE, August 2012.

UJI ANTIMITOSIS EKSTRAK ETANOL BUAH MERAH (Pandanus conoideus Lam) DENGAN METODE PENGHAMBATAN PEMBELAHAN SEL TELUR Tripneustes gratilla Linn TERFERTILISASI

The research concerning antimitotic assay of ”Buah Merah ” (Pandanus conoideus Lam ) ethanol extract using sea urchin Tripneustes gratilla Linn eggs cleavage inhibition method had been conducted. The aim of this study was to determine the antimitotic effect of buahMerah ethanol extract in to inhibit cell cleavage of fertilized sea urchin eggs. Buah Merah ethanol extract was made in to 5 concentration there was 0,001 µg/ml, 0,01 µg/ml, 0,1 µg/ml, 1 µg/ml and 10 µg/ml with vincristine as the compared. Quantivication of cell cleavage was performed visually under the light microscopy by counting the number of cleaveaged and non cleaveaged embryos after an incubation time of ± 2 hours. If 80 - 100% inhibition of cleavage (IC 80-100 ) occurs at ≤ 16 mg/ml, the compound is considered to be active as antimitotic. The result of this research showed that IC 80 value of ”BuahMerah ” ethanol extract was 0,026 mg/ml. According to the value ethanol extract was categorized active as antimitotic, although their potency lower than vincristin which can inhibit 80 % of fertilized sea urchin eggs at 2,789x10 -4 mg/ml. Key words: Pandanus conoideus Lam , A ntimitotic , Tripneustes gratilla Linn.

Isolation of C11 Compounds and a Cyclopropane Fatty Acid from an Okinawan Ascidian, Diplosoma sp.

Pentylphenols 1 and 2, cyclopropane fatty acid 3, and cyclopentenones 4 and 5, were isolated from an ascidian, Diplosoma sp. The structures of 1−5 were determined by spectroscopic analysis and/or synthesis. Compound 1 inhibited the division of fertilized sea urchin eggs and compound 4 showed mild cytotoxity against HCT116 cells (human colorectal cancer cell).

The Ecto-enzyme CD38 Is a Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Synthase That Couples Receptor Activation to Ca2+ Mobilization from Lysosomes in Pancreatic Acinar Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca(2+)-mobilizing intracellular messenger and is linked to a variety of stimuli and cell surface receptors. However, the enzyme responsible for endogenous NAADP synthesis in vivo is unknown, and it has been proposed that another enzyme differing from ADP-ribosyl cyclase family members may exist. The ecto-enzyme CD38, involved in many functions as diverse as cell proliferation and social behavior, represents an important alternative. In pancreatic acinar cells, the hormone cholecystokinin (CCK) stimulates NAADP production evoking Ca(2+) signals by discharging acidic Ca(2+) stores and leading to digestive enzyme secretion. From cells derived from CD38(-/-) mice, we provide the first physiological evidence that CD38 is required for endogenous NAADP generation in response to CCK stimulation. Furthermore, CD38 expression in CD38-deficient pancreatic AR42J cells remodels Ca(2+)-signaling pathways in these cells by restoring Ca(2+) mobilization from lysosomes during CCK-induced Ca(2+) signaling. In agreement with an intracellular site for messenger synthesis, we found that CD38 is expressed in endosomes. These CD38-containing vesicles, likely of endosomal origin, appear to be proximal to lysosomes but not co-localized with them. We propose that CD38 is an NAADP synthase required for coupling receptor activation to NAADP-mediated Ca(2+) release from lysosomal stores in pancreatic acinar cells.

UJI ANTIMITOSIS EKSTRAK ETANOL DAUN SISIK NAGA (Drymoglossum piloselloides [L.] Presl) DENGAN METODE PENGHAMBATAN PEMBELAHAN SEL TELUR Tripneustes gratilla Linn TERFERTILISASI

The research concerning antimitotic assay of “Sisik Naga” (Drymoglossum piloselloides [L] Presl) ethanol extract using Tripneustes gratilla Linn eggs cleavage inhibition method had been conducted. The aim of this study was to evaluate which the extract of that provide higher effect in to inhibit cell division of fertilized sea urchin eggs. Quantivication of division was performed visually by light microscopy by counting the number of cleaveaged and non cleaveaged embryos after an incubation time of ± 2 hours. If 80 – 100% inhibition of cleavage (IC 80-100 ) occurs at ≤ 16 mg/ml, the compound is considered to be active as antimitotic assay. Key word : Antimitotic, Drymoglossum piloselloides, vinkristin

EIF4E‐Binding proteins are differentially modified after ammonia versus intracellular calcium activation of sea urchin unfertilized eggs

Fertilization of sea urchin eggs triggers a rise of protein synthesis mainly dependent on the cap-binding protein eIF4E, which is released from its repressor 4E-BP and associates with eIF4G. Association of eIF4G with eIF4E is a crucial event for the onset of the first mitotic division following fertilization. Artificial activation of unfertilized eggs with the calcium ionophore A23187 results in the activation of protein synthesis comparable to the one triggered by fertilization, while increasing the intracellular pH by ammonia treatment results in partial activation of protein synthesis. Nevertheless, artificial activation does not induce the mitotic division. Here we investigate the effect of calcium ionophore and ammonia treatment of unfertilized eggs on eIF4E and its two antagonist partners, 4E-BP and eIF4G. We show that the addition of calcium ionophore to unfertilized eggs induces permanent dissociation between eIF4E and 4E-BP, whereas a reversible dissociation of the complex occurs after ammonia treatment. The regulation of the complex correlates with permanent or reversible 4E-BP disappearance depending on the treatment used to trigger artificial activation. Furthermore, while calcium ionophore treatment of unfertilized eggs induces eIF4G modifications comparable to those observed following fertilization, ammonia treatment does not. These results suggest that ionophore and ammonia treatments of unfertilized eggs induce differential protein synthesis activation by targeting eIF4E availability and specific regulation through its two partners 4E-BP and eIF4G.

Single-Molecule Sensitivity

Signal Transduction The external fertilization of sea urchin eggs relies on an extreme sensitivity of the sperm to chemoattractant molecules that guide the sperm toward the egg. In fact, Arbacia punctulata sperm can sense a single molecule of chemoattractant. Bonigk et al. show that such

The inhibitory action of kohamaic acid A derivatives on mammalian DNA polymerase beta.

We previously isolated a novel natural product, designated kohamaic acid A (KA-A, compound 1), as an inhibitor of the first cleavage of fertilized sea urchin eggs, and found that this compound could selectively inhibit the activities of mammalian DNA polymerases (pols). In this paper, we investigated the structure and bioactivity of KA-A and its chemically synthesized 11 derivatives (i.e., compounds 2–12), including KA-A - fatty acid conjugates. The pol inhibitory activity of compound 11 [(1S*,4aS*,8aS*)-17-(1,4,4a,5,6,7,8,8a-octahydro-2,5,5,8a-tetramethyl-naphthalen-1-yl)heptadecanoic acid] was the strongest among the synthesized compounds, and the range of IC50 values for mammalian pols was 3.22 to 8.76 μM; therefore, the length of the fatty acid side chain group of KA-A is important for pol inhibition. KA-A derivatives could prevent human cancer cell (promyelocytic leukemia cell line, HL-60) growth with the same tendency as the inhibition of mammalian pols. Since pol β is the smallest molecule, we used it to analyze the biochemical relationship with KA-A derivatives. From computer modeling analysis (i.e., docking simulation analysis), these compounds bound selectively to four amino acid residues (Leu11, Lys35, His51 and Thr79) of the N-terminal 8-kDa domain of pol β, and the binding energy between compound 11 and pol β was largest in the synthesized compounds. The relationship between the three-dimensional molecular structures of KA-A-related compounds and these inhibitory activities is discussed.

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.

Nitric oxide (NO) increase at fertilization in sea urchin eggs upregulates fertilization envelope hardening

Previous studies indicate that the nitric oxide (NO) increase at fertilization in sea urchin eggs is Ca2+-dependent and attributed to the late Ca2+ rise. However, its role in fertilization still remains unclear. Simultaneous measurements of the activation current, by a single electrode voltage clamp, and NO, using the NO indicator DAF-FM, showed that the NO increase occurred at the time of peak current (tp) which corresponds to peak [Ca2+]i, suggesting that NO is not related to any other ionic changes besides [Ca2+]i. We measured O2 consumption by a polarographic method to examine whether NO regulated a respiratory burst for protection as reported in other biological systems. Our results suggested NO increased O2 consumption. The fluorescence of reduced pyridine nucleotides, NAD(P)H was measured in controls and when the NO increase was eliminated by PTIO, a NO scavenger. Surprisingly, PTIO decreased the rate of the fluorescence change and the late phase of increase in NAD(P)H was eliminated. PTIO also suppressed the production of H2O2 and caused weak and high fertilization envelope (FE). Our results suggest that NO increase upregulates NAD(P)H and H2O2 production and consolidates FE hardening by H2O2.

Steroidal Triglycosides, Kurilensosides A, B, and C, and Other Polar Steroids from the Far Eastern Starfish Hippasteria kurilensis

Three novel steroidal triglycosides, designated as kurilensosides A, B, and C (1- 3), were isolated along with a new steroidal diglycoside, kurilensoside D (4), and two new (6, 7) and one known (5) polyhydroxysteroid from the alcoholic extract of the Far Eastern starfish Hippasteria kurilensis. Compounds 1-3 are the first triglycosides containing two carbohydrate chains found from starfish. The structures of 1-7 were elucidated by spectroscopic methods (mainly 2D NMR) and chemical derivatization. Glycosides 1-4 and steroids 6 and 7 inhibited sea urchin egg fertilization by sperm preincubated with these compounds.

An Unusual Iodinated 5′-Deoxyxylofuranosyl Nucleoside from an Okinawan Ascidian, Diplosoma sp.

Abstract An unusual nucleoside, 4-amino-7-(5′-deoxy-β-d-xylofuranosyl)-5-iodopyrrolo[2,3-d]pyrimidine (1), was isolated from an ascidian, Diplosoma sp., and its structure was successfully determined by spectroscopic and chemical analysis. The known didemnenones 2–5 were also isolated. Compound 1 was found to inhibit the division of fertilized sea urchin eggs.