Fertilized Sea Urchin Eggs Research Articles

Synthetic Studies of Furanosesquiterpenoid Tetrahydrolinderazulenes. Total Synthesis of (.+‐.)‐Echinofuran.

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The NO Pathway Acts Late during the Fertilization Response in Sea Urchin Eggs

Both the inositol 1,4,5-trisphosphate (InsP(3)) and ryanodine receptor pathways contribute to the Ca(2+) transient at fertilization in sea urchin eggs. To date, the precise contribution of each pathway has been difficult to ascertain. Evidence has accumulated to suggest that the InsP(3) receptor pathway has a primary role in causing Ca(2+) release and egg activation. However, this was recently called into question by a report implicating NO as the primary egg activator. In the present study we pursue the hypothesis that NO is a primary egg activator in sea urchin eggs and build on previous findings that an NO/cGMP/cyclic ADP-ribose (cADPR) pathway is active at fertilization in sea urchin eggs to define its role. Using a fluorescence indicator of NO levels, we have measured both NO and Ca(2+) at fertilization and establish that NO levels rise after, not before, the Ca(2+) wave is initiated and that this rise is Ca(2+)-dependent. By inhibiting the increase in NO at fertilization, we find not that the Ca(2+) transient is abolished but that the duration of the transient is significantly reduced. The latency and rise time of the transient are unaffected. This effect is mirrored by the inhibition of cGMP and cADPR signaling in sea urchin eggs at fertilization. We establish that cADPR is generated at fertilization, at a time comparable to the time of the rise in NO levels. We conclude that NO is unlikely to be a primary egg activator but, rather, acts after the initiation of the Ca(2+) wave to regulate the duration of the fertilization Ca(2+) transient.

Bioactive phloroglucinols from the brown alga Zonaria diesingiana

Three phloroglucinols with a C-20 acyl side chain were isolated from marine brown alga Zonaria diesingiana. The structures were determined on the basis of NMR spectral analyses and comparison with data in the literature. They all showed antibacterial activity against Bacillus subtilis and Staphylococcus aureus and cytotoxic activity by inhibiting cell division in fertilized sea urchin eggs (Echinometra mathaei). These activities suggest their possible for pharmacological purposes. These phloroglucinols also showed toxicity against brine shrimp (Artemia salina), guppy fish (Poecilia reticulata), rice-land shrimp (Macrobrachium lanchesteri) and the diatom Chaetoceros gracilis. They probably act as chemical defenses against herbivores and also reduce surface fouling by epiphytic algae and larvae, suggesting their important roles in the marine ecosystem.

Synthetic studies of furanosesquiterpenoid tetrahydrolinderazulenes. Total synthesis of (±)-echinofuran

Echinofuran ( 1 ) was isolated in 1992 and was reported to inhibit cell division of fertilized sea urchin eggs. The synthesis of 1 was achieved by a Ring A→Ring AC→Ring ABC approach employing 3-methyl-4-(trimethylsilyl)furan ( 2 ) as a precursor. A Suzuki coupling reaction and a Lewis acid mediated Friedel-Crafts cyclization were the other key steps in the construction of the ring systems. In other preliminary model studies, two furan-containing 5,7,5-fused tricyclic molecules were also realized.

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. α, β, γ, δ and ε) 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 μM and those of sea urchin at 6.1–30.3 μM. In the sea urchin DNA polymerases, the activities of the replicative DNA polymerases such as α, δ and ε were more strongly inhibited than that of the repair-related pol. β. 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.

Happily at Work

Happily at Work

Cytotoxic activity of chalcones isolated from lonchocarpus sericeus (pocr.) kunth.

In the present study, it was demonstrated that the hexanic extract obtained from the roots of Lonchocarpus sericeus and one of its major components, derricin, but not lonchocarpin, show cytotoxic activity to fertilized sea urchin eggs. Both inhibited the first cleavage of sea urchin eggs in a dose-dependent manner with an IC(50) of 30.4 (26.2-35.3) microgram/mL for the crude extract and 51.2 (42.1-62.3) microgram/mL for derricin (n = 6, in both cases). Furthermore, the hexanic extract of L. sericeus, and the isolated compounds, derricin and lonchocarpin showed cytotoxicity against CEM leukaemic cell line (IC(50) = 17.6 (13.7-22.6), 13.0 (12.0-14.0) and 10.4 (5.6-19.1) microgram/ml (n = 6), respectively). When these substances (6.25 to 125 microgram/25 microL) were examined for antimicrobial activity against Escherichia coli, Staphylococcus aureus and Candida albicans, none of them were found to be active. Neither the hexanic extract, nor the isolated compounds derricin and lonchocarpin (0.5 to 250 microgram/mL) presented hemolytic activity. These results indicated a possible antimitotic activity of L. sericeus crude extract and their major constituents.

Starting Out with a New Second Messenger

Churchill et al . put the final piece into place to designate nicotinic acid adenine dinucleotide phosphate (NAADP) as a second messenger. Although the Ca 2+ -mobilizing agent NAADP fulfills many criteria for a second messenger, its concentration inside cells has never been shown to increase after a physiologically relevant stimulus. Churchill et al . investigated the role of NAADP in sea urchin egg fertilization. The authors used a radioreceptor assay to demonstrate a biphasic increase in NAADP in extracts of eggs and sperm after fertilization. An increase in NAADP similar in magnitude to the earlier peak occurred in sperm incubated with egg jelly, indicating that this increase was due to NAADP production in sperm. Fertilization depleted NAADP-sensitive Ca 2+ stores in egg homogenates, but not Ca 2+ stores mobilized by inositol-1,4,5-trisphosphate (IP 3 ) or cyclic adenosine diphosphate ribose (cADPR). Intra-oocyte photorelease of NAADP--but not IP 3 , Ca 2+ , or cADPR--elicited an increase in Ca 2+ throughout the egg cortex (a cortical flash) that depended on Ca 2+ influx, as is seen after fertilization. Repeated release of NAADP led to desensitization of the NAADP-triggered cortical flash and also inhibited the cortical flash to fertilization. These data suggest that exposure to the egg elicits NAADP production by the sperm, which then introduces the NAADP into the egg, triggering the cortical flash. The increase in NAADP after fertilization, combined with the similarity in the response of the egg to NAADP to the sequelae of fertilization, establish NAADP as a bona fide second messenger in the sea urchin oocyte. G. C. Churchill, J. S. O'Neill, R. Masgrau, S. Patel, J. M. Thomas, A. A. Genazzani, A. Galione, Sperm deliver a new second messenger: NAADP. Curr. Biol. 13 , 125-128 (2003). [Online Journal]

Influence of the activator of transcription GAL4 on growth and development of embryos and embryonic cells in primary cultures of sand dollar

In order to solve many tasks of biotechnology, constant lines of the cells of marine invertebrates with a high growth potential are required, which are absent at present. We used the universal activator of transcription gal4 to change the degree of expression of genes of growth factors in embryonic sea urchin cells and, thereby, increase their proliferative activity. The fertilized sea urchin eggs and dissociated embryonic cells at the blastula stage were treated with plasmids containing both the functional gene gal4 and the gene devoid of the regions encoding the activator domain. The transfection of embryonic sea urchin eggs with the functional gene led to cell dedifferentiation and formation of tumor-like structures in the embryos or increased number of embryonic cells in culture. In the cells obtained from the transfected embryos, the pigments were found within two months of cultivation, whose absorption spectrum coincided with that of echinochrome.

Sperm Deliver a New Second Messenger: NAADP

NAADP is a highly potent mobilizer of Ca2+[1, 2], which in turn triggers Ca2+-induced Ca2+ release pathways [3–6] in a wide range of species [2, 7]. Nevertheless, NAADP is not presently classified as a second messenger because it has not been shown to increase in response to a physiological stimulus. We now report a dramatic increase in NAADP during sea urchin egg fertilization that was largely due to production in sperm upon contacting egg jelly. The NAADP bolus plays a physiological role upon delivery to the egg based on its ability to induce a cortical flash, a depolarization-induced activation of L-type Ca2+ channels. Moreover, the sperm-induced cortical flash was eliminated in eggs desensitized to NAADP. We conclude that an NAADP increase plays a physiologically relevant role during fertilization and provides the first conclusive demonstration that NAADP is a genuine second messenger.

Utilization of a particle gun DNA introduction system for the analysis of cis-regulatory elements controlling the spatial expression pattern of the arylsulfatase gene (HpArs) in sea urchin embryos.

We applied a particle gun method to introduce DNA into fertilized sea urchin eggs for the analysis of cis-regulatory elements responsible for spatial gene expression during development. We introduced HpArs (sea urchin arylsulfatase gene) -GFP and HpArs-LacZfusion constructs into the fertilized eggs and obtained high expression levels of the fusion genes. Using this assay system, we demonstrated that a fragment of HpArs (-3,484 to +4,636) is sufficient for aboral ectoderm-specific expression, and that the region in the first intron from +406 to +1,993 contains the control elements responsible for the repression of the HpArs promoter activity in secondary mesenchyme cells.

Two new minor polybrominated dibenzo-p-dioxins from the marine sponge Dysidea dendyi.

Two new minor tribromodibenzo-p-dioxins, spongiadioxin C (1) and its methyl ether (2), were isolated from an Australian marine sponge Dysidea dendyi, together with the known minor metabolites methyl ethers of spongiadioxins A (4) and B (6) and polybrominated diphenyl ethers (7-9). The structures of the new compounds were established by 1D and 2D NMR spectroscopy and confirmed by synthesis of 2 from diphenyl ether 9. All isolated compounds inhibited the cell division of fertilized sea urchin eggs.

Increase of cGMP, cADP-ribose and inositol 1,4,5-trisphosphate preceding Ca(2+) transients in fertilization of sea urchin eggs.

Transient increases, or oscillations, of cytoplasmic free Ca(2+) concentration, [Ca(2+)](i), occur during fertilization of animal egg cells. In sea urchin eggs, the increased Ca(2+) is derived from intracellular stores, but the principal signaling and release system involved has not yet been agreed upon. Possible candidates are the inositol 1,4,5-trisphosphate receptor/channel (IP(3)R) and the ryanodine receptor/channel (RyR) which is activated by cGMP or cyclic ADP-ribose (cADPR). Thus, it seemed that direct measurements of the likely second messenger candidates during sea urchin fertilization would be essential to an understanding of the Ca(2+) signaling pathway. We therefore measured the cGMP, cADPR and inositol 1,4,5-trisphosphate (IP(3)) contents of sea urchin eggs during the early stages of fertilization and compared these with the [Ca(2+)](i) rise in the presence or absence of an inhibitor against soluble guanylate cyclase. We obtained three major experimental results: (1) cytosolic cGMP levels began to rise first, followed by cADPR and IP(3) levels, all almost doubling before the explosive increase of [Ca(2+)](i); (2) most of the rise in IP(3) occurred after the Ca(2+) peak; IP(3) production could also be induced by the artificial elevation of [Ca(2+)](i), suggesting the large increase in IP(3) is a consequence, rather than a cause, of the Ca(2+) transient; (3) the measured increase in cGMP was produced by the soluble guanylate cyclase of eggs, and inhibition of soluble guanylate cyclase of eggs diminished the production of both cADPR and IP(3) and the [Ca(2+)](i) increase without the delay of Ca(2+) transients. Taken together, these results suggest that the RyR pathway involving cGMP and cADPR is not solely responsible for the initiating event, but contributes to the Ca(2+) transients by stimulating IP(3) production during fertilization of sea urchin eggs.

Cacofurans A and B, new furanoditerpenes from a marine sponge.

Two new labdane-class diterpenes, cacofurans A (1) and B (2), have been isolated from a sponge Cacospongia sp. Their structures were determined by analyzing spectroscopic data, by chemical transformations, and by X-ray diffraction. Cacofurans 1 and 2 inhibited the development of fertilized sea urchin eggs at concentrations of 0.5 and 5 microg/mL and showed an actin-disrupting effect on the NBT-II cell line at 10 microg/mL.

Fertilization of Sea Urchin Eggs and Sperm Motility Are Negatively Impacted under Low Hypergravitational Forces Significant to Space Flight1

Sperm and other flagellates swim faster in microgravity (microG) than in 1 G, raising the question of whether fertilization is altered under conditions of space travel. Such alterations have implications for reproduction of plant and animal food and for long-term space habitation by man. We previously demonstrated that microG accelerates protein phosphorylation during initiation of sperm motility but delays the sperm response to the egg chemotactic factor, speract. Thus sperm are sensitive to changes in gravitational force. New experiments using the NiZeMi centrifugal microscope examined whether low hypergravity (hyperG) causes effects opposite to microG on sperm motility, signal transduction, and fertilization. Sperm % motility and straight-line velocity were significantly inhibited by as little as 1.3 G. The phosphorylation states of FP130, an axonemal phosphoprotein, and FP160, a cAMP-dependent salt-extractable flagellar protein, both coupled to motility activation, showed a more rapid decline in hyperG. Most critically, hyperG caused an approximately 50% reduction in both the rate of sperm-egg binding and fertilization. The similar extent of inhibition of both fertilization parameters in hyperG suggests that the primary effect is on sperm rather than eggs. These results not only support our earlier microG data demonstrating that sperm are sensitive to small changes in gravitational forces but more importantly now show that this sensitivity affects the ability of sperm to fertilize eggs. Thus, more detailed studies on the impact of space flight on development should include studies of sperm function and fertilization.

Measurement of the intracellular pH threshold for sperm aster formation in sea urchin eggs.

In the fertilization of sea urchin eggs, intracellular [Ca2+] (Cai) increases transiently and intracellular pH (pHi) elevates accordingly. Unlinking these two activating factors experimentally, the requirement of the increase in pHi for sperm aster formation in the sea urchin, Clypeaster japonicus, was investigated. When the eggs were injected with an EGTA or BAPTA solution, they incorporated sperm but did not organize the sperm aster. Using these sperm-incorporated eggs under the condition that an increase in Cai was blocked, pHi was regulated by two methods: (i) perfusing ammonium acetate-containing seawater; and (ii) injecting pH buffer solutions of various pH values. By either of the two methods, the sperm aster formed at pHi 7.0 or more and functioned in female pronuclear migration when the sperm aster reached the female pronucleus. Hence, the step of the transient increase in Cai at fertilization can be bypassed. In contrast, a pHi increase is indispensably required for sperm aster formation in sea urchin eggs. Moreover, under the condition that there was the transient increase in Cai, the threshold pHi value for sperm aster formation was pHi 7.0 or more. Consequently, whether a Cai increase on fertilization occurs or not, the threshold pHi value for sperm aster formation is constant in sea urchin eggs.

Calcium-Mediated Inactivation of the MAP Kinase Pathway in Sea Urchin Eggs at Fertilization

We have evaluated the regulation of a 43-kDa MAP kinase in sea urchin eggs. Both MAP kinase and MEK (MAP kinase kinase) are phosphorylated and active in unfertilized eggs while both are dephosphorylated and inactivated after fertilization, although with distinct kinetics. Reactivation of MEK or the 43-kDa MAP kinase prior to or during the first cell division was not detected. Confocal immunolocalization microscopy revealed that phosphorylated (active) MAP kinase is present primarily in the nucleus of the unfertilized egg, with some of the phosphorylated form in the cytoplasm as well. Incubation of unfertilized eggs in the MEK inhibitor U0126 (0.5 μM) resulted in the inactivation of MEK and MAP kinase within 30 min. Incubation in low concentrations of U0126 (sufficient to inactivate MEK and MAP kinase) after fertilization had no effect on progression through the embryonic cell cycle. Microinjection of active mammalian MAP kinase phosphatase (MKP-3) resulted in inactivation of MAP kinase in unfertilized eggs, as did addition of MKP-3 to lysates of unfertilized eggs. Incubation of unfertilized eggs in the Ca2+ ionophore A23187 led to inactivation of MEK and MAP kinase with the same kinetics as observed with sperm-induced egg activation. This suggests that calcium may be deactivating MEK and/or activating a MAP kinase-directed phosphatase. A cell-free system was used to evaluate the activation of phosphatase separately from MEK inactivation. Unfertilized egg lysates were treated with U0126 to inactivate MEK and then Ca2+ was added. This resulted in increased MAP kinase phosphatase activity. Therefore, MAP kinase inactivation at fertilization in sea urchin eggs likely is the result of a combination of MEK inactivation and phosphatase activation that are directly or indirectly responsive to Ca2+.

New steroid glycosides from the starfish Asterias rathbuni.

Two novel steroidal 24-O-xylosides, designated as rathbuniosides R(1) (1) and R(2) (2), and the known amurensoside A (3) and 3-O-sulfomarthasterone (4) have been isolated from the starfish Asterias rathbuni. The structures of all the compounds were determined from their spectroscopic data, including one- and two-dimensional NMR methods. The compounds 1 and 4 inhibit the cell division of fertilized sea urchin eggs at doses of 7.0 x 10(-5) and 2.9 x 10(-5) M, respectively.

Structure–activity relationship for bromoindole carbaldehydes: Effects on the sea urchin embryo cell cycle

Natural derivatives of indole-3-carbaldehyde were isolated from the tropical marine ascidian Stomoza murravi. A series of 13 derivatives, three natural and 10 synthetic (brominated and N-methylated), were examined for their effects on cell division of sea urchin eggs. These derivatives were shown to inhibit the first mitotic cycle in a concentration-dependent manner. By comparing the IC50 values with the structure of the various molecules, we were able to determine that bromination increased the cytotoxicity of the compound with a maximum occurring when bromine was added to carbon number 2, while addition of N-methylation was shown to markedly reduce the cytotoxicity of these same compounds brominated at carbon 2 only. Biological activity of this family of compounds has been characterized, via detailed study of addition of the most active derivative, 2,5,6-tribromoindole-3-carbaldehyde, on macromolecule synthesis and cytoskeleton reorganization during the first mitotic cycle of fertilized sea urchin eggs. Fluorescence localization of chromatin and microtubules revealed that 2,5,6-tribromoindole-3-carbaldehyde allowed pronuclei migration and fusion but prevented the condensation of chromatin, nuclear envelope breakdown, and bipolar mitotic spindle assembly, inducing an arrest of sea urchin embryogenesis at the beginning of mitosis. It is postulated here that this phenotype is likely to be due to a strong inhibition of DNA replication and protein synthesis.

Effects of gamete dilution, age and contact time on fertilization success in the tropical sea urchin, Echinometra mathaei

A series of laboratory experiments were conducted to determine the effect of sperm dilution, egg concentration, sperm-egg contact time, and gamete aging on fertilization success in the tropical sea urchin, Echinometra mathaei. The results demonstrated that sperm dilution, sperm age, and sperm-egg contact time were sequentially the most important factors influencing fertilization success, while egg concentration was not significant over the range tested. Sperms retained their potency for more than two hours only in relatively dense sperm suspensions (≥10-4 dilution of ‘dry’ sperm) whereas they exhibited lower viability with increasing dilutions and age. In egg-sperm contact time experiments more than 80% fertilization was achieved at lower sperm dilutions (10-3-10-2) within 10 sec of mixing, while at higher sperm dilutions, longer times of contact were needed to achieve the similar fertilizations. Consequently, eggs remained in good quality for up to 3 h and there was no abnormality or adverse effects in fertilization were observed in a series of sperm dilution tested. These laboratory experiments suggest that sperm dilution and its limited longevity can play an important role in limiting the fertilization of sea urchin eggs in the field during natural spawnings. It follows, therefore, that sea urchin (E. mathaei) are under considerable selective pressures to spawn synchronously in order to generate high sperm concentrations and higher sperm-egg encounters in the water column to maximize the probability of successful fertilization.