Calcium-free Seawater Research Articles

Rho-family G-proteins are required for the recovery of traumatized hair bundle mechanoreceptors in the sea anemone, Nematostella vectensis

Immersing anemones in calcium-free seawater disorganizes hair bundle mechanoreceptors on tentacles of sea anemones while causing a loss of vibration sensitivity. Remarkably, anemone hair bundles recover after being returned to calcium-containing seawater. Reorganization of actin in stereocilia likely follows during the recovery of normal morphology of hair bundles after such immersion. Previous studies have reported that Rho G-proteins are located in the stereocilia of hair bundles in sea anemones where they participate in polymerizing actin in stereocilia upon activation of specific chemoreceptors. We here find that immersing anemones in calcium-free seawater significantly reduces the abundance of hair bundles. A partial recovery of abundance of hair bundles occurs within 3 h post-immersion, but a full recovery of abundance does not occur even 6 h after specimens are returned to calcium-containing seawater. Anemones recovering from immersion in calcium-free seawater feature hair bundles that are significantly wider at their tips than in controls. The hair bundles subsequently narrow at their tips, becoming comparable to those of untreated controls within 6 h. Stereocilia of hair bundles are significantly longer in experimental animals than in controls at 2 h of recovery before shortening to lengths comparable to untreated controls at 6 h. In the presence of Rho inhibitors, the recovery in abundance of hair bundles through 6 h is delayed or inhibited. Likewise, in the presence of Rho inhibitors, stereocilia fail to significantly elongate within 2 h of recovery. These data suggest that Rho G-proteins participate in the normal recovery of abundance and recovery of normal morphology of experimentally damaged hair bundle mechanoreceptors.

Novel Ca2+ increases in the maturing oocytes of starfish during the germinal vesicle breakdown

It has been known that the intracellular Ca2+ level transiently rises at the specific stages of mitosis such as the moment of nuclear envelope breakdown and at the metaphase–anaphase transition. Comparable intracellular Ca2+ increases may also take place during meiosis, as was intermittently reported in mouse, Xenopus, and starfish oocytes. In a majority of starfish species, the maturing oocytes display an intracellular Ca2+ increase within few minutes after the addition of the maturation hormone, 1-methyladenine (1-MA). Although starfish oocytes at meiosis also manifest a Ca2+ increase at the time of polar body extrusion, a similar Ca2+ increase has never been observed during the envelope breakdown of the nucleus (germinal vesicle, GV). Here, we report, for the first time, the existence of an additional Ca2+ response in the maturing oocytes of Asterina pectinifera at the time of GV breakdown. In contrast to the immediate early Ca2+ response to 1-MA, which is independent of external Ca2+ and takes a form of intracellular Ca2+ wave traveling three times as fast as that in the fertilized eggs, this late stage Ca2+ response comprised a train of numerous spikes representing Ca2+ influx. These Ca2+ spikes coinciding with GV breakdown were mostly eliminated when the GV was removed from the oocytes prior to the addition of 1-MA, suggesting that the Ca2+ spikes are rather a consequence of the GV breakdown. In support of the idea that these Ca2+ spikes play a physiological role, the oocytes matured in calcium-free seawater had a higher rate of cleavage failure 2h after the fertilization in natural seawater. Specific inhibitors of L-type Ca2+ channels, verapamil and diltiazem, severely suppressed the amplitude of the individual Ca2+ spikes, but not their frequencies. On the other hand, latrunculin-A (LAT-A), which promotes net depolymerization of the actin cytoskeleton, had a dual effect on this late Ca2+ response. When added immediately after the hormone-dependent period, LAT-A inhibited the occurrence (frequency) of the spikes in a dose-dependent manner, but the amplitude of the prevailing Ca2+ spikes itself was rather significantly increased. These results suggest that the cortical actin cytoskeleton and some nuclear factors may play a role in regulating ion channel activities during this stage of meiotic progression.

The B vitamins nicotinamide (B3) and riboflavin (B2) stimulate metamorphosis in larvae of the deposit-feeding polychaete Capitella teleta: implications for a sensory ligand-gated ion channel.

Marine sediments can contain B vitamins, presumably incorporated from settled, decaying phytoplankton and microorganisms associated with decomposition. Because B vitamins may be advantageous for the energetically intensive processes of metamorphosis, post-metamorphic growth, and reproduction, we tested several B vitamins to determine if they would stimulate larvae of the deposit-feeding polychaete Capitella teleta to settle and metamorphose. Nicotinamide and riboflavin individually stimulated larvae of C. teleta to settle and metamorphose, generally within 1–2 hours at nicotinamide concentrations as low as 3 µM and riboflavin concentrations as low as 50 µM. More than 80% of the larvae metamorphosed within 30 minutes at a nicotinamide concentration of 7 µM. The pyridine channel agonist pyrazinecarboxamide also stimulated metamorphosis at very low concentrations. In contrast, neither lumichrome, thiamine HCl, pyridoxine HCl, nor vitamin B12 stimulated larvae of C. teleta to metamorphose at concentrations as high as 500 µM. Larvae also did not metamorphose in response to either nicotinamide or pyrazinecarboxamide in calcium-free seawater or with the addition of 4-acetylpyridine, a competitive inhibitor of the pyridine receptor. Together, these results suggest that larvae of C. teleta are responding to nicotinamide and riboflavin via a chemosensory pyridine receptor similar to that previously reported to be present on crayfish chela and involved with food recognition. Our data are the first to implicate B vitamins as possible natural chemical settlement cues for marine invertebrate larvae.

Signaling pathways in ascidian oocyte maturation: The roles of cAMP/Epac, intracellular calcium levels, and calmodulin kinase in regulating GVBD

Most mature ascidian oocytes undergo germinal vesicle breakdown (GVBD) when released by the ovary into sea water (SW). Acidic SW blocks this but they can be stimulated by raising the pH, increasing intracellular cAMP levels by cell permeant forms, inhibiting its breakdown or causing synthesis. Boltenia villosa oocytes undergo GVBD in response to these drugs. However, the cAMP receptor protein kinase A (PKA) does not appear to be involved, as oocytes are not affected by the kinase inhibitor H-89. Also, the PKA independent Epac agonist 8CPT-2Me-cAMP stimulates GVBD in acidic SW. GVBD is inhibited in calcium free sea water (CaFSW). The intracellular calcium chelator BAPTA-AM blocks GVBD at 10 µM. GVBD is also inhibited when the ryanodine receptors (RYR) are blocked by tetracaine or ruthenium red but not by the IP(3) inhibitor D-609. However, dimethylbenzanthracene (DMBA), a protein kinase activator, stimulates GVBD in BAPTA, tetracaine or ruthenium red blocked oocytes. The calmodulin kinase inhibitor KN-93 blocks GVBD at 10 µM. This and preceding papers support the hypothesis that the maturation inducing substance (MIS) produced by the follicle cells in response to increased pH causes activation of a G protein which triggers cAMP synthesis. The cAMP then activates an Epac molecule, which causes an increase in intracellular calcium from the endoplasmic reticulum ryanodine receptor. The increased intracellular calcium subsequently activates calmodulin kinase, which causes an increase in cdc25 phosphatase activity, activating MPF and the progression of the oocyte into meiosis.

Stimulators of AMP‐activated kinase (AMPK) inhibit seawater‐ but not cAMP‐induced oocyte maturation in a marine worm: Implications for interactions between cAMP and AMPK signaling

Previous studies have shown that elevations in intraoocytic cAMP prevent mammalian oocytes from maturing, whereas cAMP degradation allows these oocytes to begin maturation, as evidenced by the onset of oocyte nuclear disassembly (="germinal vesicle breakdown", GVBD). Moreover, such cAMP degradation not only reduces cAMP levels but also generates AMP, which in turn can stimulate AMP-activated kinase (AMPK), a well-documented inducer of GVBD in mice. Alternatively, in some marine invertebrates, intraoocytic cAMP triggers, rather than blocks, GVBD, and whether AMPK up- or downregulates maturation in these species has not been tested. Thus, AMPK was monitored in the nemertean worm Cerebratulus during GVBD stimulated by seawater (SW) or cAMP elevators. In oocytes lacking surrounding follicle cells, AMPK activity was initially elevated in immature oocytes but subsequently reduced during SW- or cAMP-induced GVBD, given that the catalytic alpha-subunit of AMPK in maturing oocytes displayed a decreased stimulatory phosphorylation at T172 and an increased inhibitory phosphorylation at S485/491. Accordingly, AMPK-mediated phosphorylation of acetyl-CoA carboxylase, a known target of active AMPK, also declined during maturation. Moreover, treatments with either ice-cold calcium-free seawater (CaFSW) or AMPK agonists dissolved in SW maintained AMPK activity and inhibited GVBD. Conversely, adding cAMP elevators to CaFSW- or SW-solutions of AMPK activators restored GVBD while promoting S485/491 phosphorylation and AMPK deactivation. Collectively, such findings not only demonstrate for the first time that intraoocytic AMPK can block GVBD in the absence of surrounding follicle cells, but these results also provide evidence for a novel GVBD-regulating mechanism involving AMPK deactivation by cAMP-mediated S485/491 phosphorylation.

Pre‐ and postsynaptic excitation and inhibition at octopus optic lobe photoreceptor terminals; implications for the function of the ‘presynaptic bags’

Synaptic transmission was examined in the plexiform zone of Octopus vulgaris optic lobes using field-potential recording from optic lobe slices. Stimulation of the optic nerve produced pre- and postsynaptic field potentials. Transmission was abolished in calcium-free seawater, L- glutamate or the AMPA/Kainate receptor blocker CNQX (EC(50), 40 microm), leaving an intact presynaptic field potential. ACh markedly reduced or blocked and d-tubocurarine augmented both pre- and postsynaptic field potentials, while alpha-bungarotoxin and atropine were without effect. Paired-pulse stimulation showed short-term depression of pre- and postsynaptic components with a half-time of recovery of approximately 500 ms. The depression was partially relieved in the presence of d-tubocurarine (half-time of recovery, 350 ms). No long-term changes in synaptic strength were induced by repetitive stimulation. A polyclonal antibody raised against a squid glutamate receptor produced positive staining in the third radial layer of the plexiform zone. No positive staining was observed in the other layers. Taking into account previous morphological data and our results, we propose that the excitatory terminations of the photoreceptors are in the innermost layer of the plexiform zone where the transmitter is likely to be glutamate and postsynaptic receptors are AMPA/kainate-like. Thus, the function of the terminal bags is to provide a location for a presynaptic cholinergic inhibitory shunt. The results imply that this arrangement provides a temporal filter for visual processing and enhances the perception of moving vs. stationary objects.

Do heavy metals play an active role in sponge cell behaviour in the absence of calcium? Consequences in larval settlement

Moderate concentrations of copper and cadmium can have positive effects on sponges by enhancing cell aggregation and larval settlement. The cellular events underlying these effects are not fully understood, but it is known that cell aggregation needs of an extracellular supply of calcium, which in the absence of external calcium may also be provided by the release of internal calcium stores. In order to know the mechanisms by which Cd 2+ and Cu 2+ affected sponge cells and larvae and whether the alteration of calcium homeostasis is involved in such effects, we incubated Scopalina lophyropoda cells for 3 h and larvae for seven days in calcium-free seawater (CFSW), seawater (SW), and CFSW with copper (Cu CFSW) and cadmium (Cd CFSW). Both metals seem to play an active role in calcium dependent processes: sponge cells incubated in Cd CFSW and Cu CFSW, aggregated as much as those in SW control, while aggregates were rare in CFSW. Similarly, Cd and Cu first fastened larval settlement but toxic effects of both metals were evidenced after a longer exposition (five days), leading to settler mortality. Thus, although the effect of these two heavy metals may be positive in the short-term, long term exposition or higher concentrations may alter cell functions and affect sponge viability and fitness.

Extracellular Ca2+ requirement for serotonin-induced release and meiosis reinitiation from prophase in oocytes of the scallop Argopecten purpuratus

Summary The requirement for Ca2+in the external medium for the release and reinitiation of meiosis in the scallop Argopecten purpuratus was examined. In vitro experiments demonstrated that the serotonin-induced release of oocytes is inhibited in a calcium-free medium. Caffeine (10 and 20 mM), a known activator of ryanodine channels, increased oocyte release by gonadal pieces and 50 μM ryanodine inhibited this release and blocked the serotonin-induced effect. The number of oocytes released under control conditions was not affected when ryanodine was used in concentrations in the nanomolar range. Germinal vesicle breakdown, indicative of meiosis reinitiation, was inhibited in gonadal pieces incubated in calcium-free normal or artificial seawater and serotonin does not overcome this inhibition in the same conditions. An increase in fluorescence on adding serotonin (10−5 M, final concentration) was detected under the confocal microscope when oocytes charged with the calcium fluorescent dye Fluo-3 AM were studied. We postulate that there is a rise in cellular calcium in the serotonin-induced spawning process and that the source for the increase is influx from the external medium and release from internal stores. We suggest that this calcium rise plays a role in germinal vesicle breakdown and in the contraction of gonadal smooth muscle.

Effects of calcium-free and low-calcium artificial seawater on polyps of a scleractinian coral Galaxea fascicularis

Investigations of various aspects of calcification incorals have frequently made use of artificial seawatersfrom which calcium has been omitted or has beenpresent in low concentrations compared to standardseawater (Chalker 1976; Krishnaveni et al. 1989; Ip andKrishnaveni 1991; Al-Moghrabi et al. 1996; Tambutteet al. 1996; Gattuso et al. 2000). The possibility ofundesirable effects resulting from exposure to suchmedia does not appear to have been considered. Thereappears to have been no attempt to investigate the non-physiological effects of calcium depletion in seawatereven though it has been long known that calcium hasprofound effects on membrane permeabilities andnumerous physiological processes (Hoar 1966; Manery1966; Giese 1979).The purpose of the present investigation was toascertain whether calcium-free seawater, or seawatercontaining low calcium concentrations, had any obviouseffects on the polyps of Galaxea fascicularis. A numberof artificial seawater formulations have been used bydifferent investigators. We report the effects of three lowcalcium or calcium-free formulations, used by previousinvestigators, and make comparisons with the corre-sponding standard artificial seawaters and naturalstandard seawater.

Multiple triggers of oocyte maturation in nemertean worms: the roles of calcium and serotonin.

To analyze the process of oocyte maturation in nemertean worms, oocytes with a large nucleus (=germinal vesicle, or GV) were removed from gravid ovaries of Cerebratulus lacteus and Micrura alaskensis. Following transfer to natural seawater (NSW), fully grown oocytes spontaneously matured as indicated by their completion of germinal vesicle breakdown (GVBD), whereas GVBD was reversibly blocked if the oocytes were initially placed in calcium-free seawater (CaFSW). Similarly, calcium ionophore treatments triggered GVBD in calcium-containing artificial seawater (ASW) but not in CaFSW, suggesting that external calcium influx may facilitate maturation. However, compared to the overall levels of maturation elicited by ASW, significantly higher percentages of GVBD were achieved with NSW or with ASW that had been conditioned with marine sediment. Moreover, calcium channel blockers decreased GVBD rates in ASW but not in NSW, which is consistent with the view that substances other than external calcium ions can trigger maturation. Accordingly, oocytes underwent equally high levels of GVBD when treated with serotonin (=5-hydroxytryptamine, or 5-HT) in ASW or CaFSW. The 5-HT-induced maturation was blocked by inhibitors of 5-HT receptors but continued to occur in the presence of calcium channel blockers or the calcium chelator BAPTA. In addition, oocytes microinjected with fluorescent calcium indicators underwent GVBD in response to 5-HT without displaying marked calcium transients during confocal imaging runs. Collectively, such findings suggest that nemertean oocytes can mature via multiple pathways that may include external calcium influx or a 5-HT-induced signaling cascade that lacks prominent calcium fluctuations. J. Exp. Zool. 287:243-261, 2000.

Competence of the animal cap to react with the inductive signal from micromere descendants in the hatching blastula stage of echinoid embryos

Micromere signalling is a key to understanding the developmental mechanisms underlying endomesoderm differentiation along the A-V axis in sea urchin embryos. A recent study has shown that micromere activity in inducing endo-mesoderm differentiation of mesomere descendants is, unexpectedly, maximal at the hatching blastula (HB) stage in the echinoids Scaphechinus mirabilis and Hemicentrotus pulcherrimus. This study focused mainly on the timing of emission of the inductive signal by the micromere descendants. The timing of animal cap competent to react to the inductive signal from micromeres was not specifically investigated.In the present study, we examined the competence of mesomere descendants at the HB stage to react to the inductive signal of micromere descendants by producing recombinant embryos of the descendants of mesomeres and micromeres.Adults of the sand dollar Scaphechinus mirabilis and the sea urchin Hemicentrotus pulcherrimus were collected along the shore of Shiraishi Island, Okayama Prefecture and in the vicinity of the Misaki Marine Biological Station, Kanagawa Prefecture, respectively. The fertilised eggs were separated into two groups immediately after removal of the fertilisation membranes. One group was cultured in normal artificial seawater (ASW), and the other in ASW containing 50 mg/ml of rhodamine B isothiocyanate (RITC). At the early 16-cell stage unlabelled embryos were transferred to calcium-free seawater (CFSW) and dissected in the equatorial plane with a glass needle to isolate animal caps consisting of eight mesomeres. Embryos labeled with RITC were also transferred to CFSW at the same stage and dissected to isolate four micromeres. An isolated animal cap and a quartet of micromeres were cultured separately in ASW and recombined at the stage corresponding to the HB of control (undisturbed) embryos.

Isolation and primary culture of viable multicellular endothelial isolates from hard corals.

Conditions for the primary culture of branching scleractinian coral (Acropora micropthalma and Pocillopora damicornis) cells were established with a calcium-free seawater cell dissociation method. Cells were isolated and cultured in supplemented Dulbecco's modified Eagle media with heat-inactivated fetal bovine serum, antibiotics, and sterile seawater. Among the isolated cell types, large (60-100 microm) multicellular endothelial isolates (MEIs) were seen in high numbers. These isolates were observed to continually spin for up to 300 h without media change. The following parameters were optimized: media, serum, light, trace elements, and growth factor supplements. Rotations per minute were calculated to determine MEI motility in relation to size. Finally, analyses of external and internal structures were conducted with scanning electron microscopy, transmission electron microscopy, and fluorescence microscopy. Additional coral species, Montipora digitata, Stylophora pistillata, Seriatopora hystrix and Porites sp. were also cultured to determine the applicability of isolation techniques. The relatively long survival time of MEIs in primary culture makes them ideal candidates for in vitro studies examining coral disease processes (e.g., mode of infection and intracellular effects of disease-causing agents) as well as aspects of general coral growth and health (e.g., trace element requirements and transfer of products between host cell and zooxanthellae).

Chemoreception in the nudibranch gastropod Phestilla sibogae

The tropical nudibranch Phestilla sibogae feeds exclusively on corals of the genus Porites, which it locates and recognizes by chemical cues. Morphological and physiological analyses of chemosensory neural pathways in P. sibogae focussed on two pairs of cephalic tentacles, the rhinophores and the oral tentacles. Two nerves from each oral tentacle pass directly to the brain, whereas multiple nerves from the rhinophores converge on paired rhinophoral ganglia that connect to the cerebral ganglia. Chemical sensitivity, determined by changes in rates of discharge, was monitored by suction electrodes attached to cut ends of rhinophoral or oral-tentacle nerves while the excised structure was perfused with extracts of Porites spp., non-food corals, l-amino acids or glutamate-receptor modulators. Although the oral tentacles were relatively insensitive to the substances tested, responses in rhinophores were positive to extracts of Porites compressa and a non-food coral, were biphasic to l-aspartic and l-glutamic acids and were perceptible but weak to several other amino acids. Rhinophores responded positively to the glutamate-receptor modulators d-glutamate, 6,7-dinitroquinoxaline-2,3-dione (DNQX) and kainic acid but not to N-methyl-D-aspartate (NMDA); the initial inceased firing-rate response to l-glutamate disappeared when the glutamate was dissolved in magnesium- or cobalt-substituted calcium-free seawater. For the substances tested here, including food corals and a set of amino acids, the rhinophores are the main chemosensory organs for P. sibogae. Glutamate receptors pharmacologically similar to the kainic acid type of vertebrates are present.

Repetitive Calcium Waves Induced by Fertilization in the Nemertean WormCerebratulus lacteus

To analyze fertilization-induced calcium dynamics in a protostome worm, unfertilized oocytes of the nemerteanCerebratulus lacteuswere co-injected with calcium green (CG) and rhodamine (Rh) dextrans for dual-channel confocal imaging of early development. Based on CG/Rh ratioed images collected every 800 msec, fertilization elicits a “cortical flash” of elevated free calcium that spreads rapidly around the oocyte without propagating as a point-source wave. A similar calcium transient occurs in unfertilized oocytes treated with KCl to depolarize the oolemma, and the fertilization-induced cortical flash is eliminated if cobalt is used to block calcium channels, collectively indicating that fertilization initially triggers an influx of calcium ions through voltage-gated calcium channels in the oolemma. However, within minutes after producing a cortical flash,C. lacteusoocytes begin to display a series of point-source, oscillating waves of elevated free calcium that are propagated at about 15 μm/sec. The first two calcium waves arise at the site of sperm fusion and typically fail to reach the antipode, but after sperm incorporation, the waves spread globally throughout the ooplasm and typically shift their origin to a pacemaker region in the vegetal cortex. About 10 oscillations with an average duration of 3.3 ± 1.2 min are generated for ∼60–100 min postfertilization as meiotic maturation is completed, and such waves continue to occur in cobalt-containing seawater or calcium-free seawater. Thus, wavelike calcium oscillations induced by fertilization are apparently dependent upon internal calcium stores, which in turn may contain IP3-insensitive and/or IP3-sensitive receptors based on experiments using ryanodine, caged IP3, and heparin. Unfertilized oocytes also display repetitive calcium waves following intracytoplasmic injections of whole sperm, and such oscillations are eliminated if the sperm suspensions are boiled prior to injection, suggesting the possible presence of a heat-labile sperm component that can elicit wavelike oscillations during fertilization.

Dye coupling in the muscles controlling squid chromatophore expansion

Dye coupling between the cone-shaped radial muscle fibres, which control the expansion and closing of a squid chromatophore organ, was investigated in the squid Loligo vulgaris. Particular attention was paid to the role of the myomuscular junctions located between the muscle fibres. Lucifer Yellow was injected ionophoretically into single muscle fibres under normal artificial sea water (ASW) and under various concentrations of calcium in ASW. Under ASW, 44% of muscle fibres examined were dye-coupled, 82% were coupled under calcium-free sea water and 67% were coupled under sea water containing high concentrations of calcium. Dye transfer was blocked by octanol. Muscle fibres were never seen to link adjacent chromatophore organs. Results are discussed in terms of the role of the myomuscular junctions in the regulation of chromatophore expansion in the living animal.

Different calcium-dependent pathways control fertilisation-triggered glycoside release and the cortical contraction in ascidian eggs.

Fertilisation of ascidian eggs induces the rapid release of a cell surface N-acetylglycosaminidase that blocks sperm binding to vitelline coat sperm receptors resulting in a block to polyspermy. Fertilisation also triggers a large contraction of the egg (thus stimulating ooplasmic segregation) that is completed within 5 min of insemination. In eggs of the ascidian Phallusia mammillata, glycosidase release and cortical contractions are blocked by BAPTA-AM [bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester], a cell-permeant calcium chelator, indicating that both processes are probably dependent on a rise in intracellular calcium levels. Both glycosidase release and the cortical contraction are induced by treatment of the egg with the protein synthesis inhibitor emetine, while only the glycosidase release is induced by isoproterenol, carbachol or acetylcholine. Previous work with ryanodine demonstrated that ryanodine also caused glycosidase release but not the cortical contraction. Inversely, activation by ionomycin in calcium-free sea water causes cortical contractions but not glycosidase release. Thus the two processes can be activated independently. Dextran-coupled (10 kDa) calcium green-1 injected eggs show an increase in intracellular calcium 30-40 s before the cortical contraction is triggered by fertilisation or ionomycin-induced activation. This confirms previous findings that the cortical contraction is a consequence of the activation calcium wave triggered by the sperm. The extracellular calcium requirement for the glycosidase release suggests that calcium influx may be more important for this phase of egg activation. Thus activation of ascidian eggs appears to involve two independent pathways involving calcium.

Calcium and Cyclic AMP Mediate Sperm Activation, but Ca2+ Alone Contributes Sperm Chemotaxis in the Ascidian, Ciona savignyi: (ascidian/sperm motility/chemotaxis/calcium/cAMP).

Sperm-activating and -attracting factor (SAAF) released from the ascidian, Ciona savignyi, was partially purified from egg seawater with ethanol extraction and separation with the two-phase system of chloroform and water. SAAF did not activate sperm motility and cAMP synthesis in calcium-free seawater (CaFSW), but activated the both in the presence of Ca2+ . Sperm activation by SAAF in Ca2+ -containing medium was inhibited by flunarizine, a T-type Ca2+ channel antagonist, but L-type Ca2+ channel specific antagonists had no effect. Theophylline, a phosphodiesterase inhibitor, induced the increase of cAMP level and sperm activation in CaFSW without SAAF. On the other hand, the theophylline-activated sperm in CaFSW did not exhibit chemotaxis toward the tip of glass capillary containing SAAF, but upon the addition of Ca2+ they were attracted toward SAAF in the same manner as chemotaxis in normal artificial seawater. These results suggest that sperm activation is induced by the increased cAMP level caused by Ca2+ influx through T-type Ca2+ channel, and that Ca2+ alone mediates the sperm chemotaxis in Ciona.

Responses of two proboscis muscles of Neptunea antiqua (mollusca) to some calcium antagonist drugs

1. Both the radular retractor (RR) and radular sac (RS) muscles of Neptunea antiqua depend upon [Ca] 0 to raise the internal calcium concentration of the contractile elements to activation level. 2. The K- and ACh-induced responses of the muscles were strongly inhibited in calcium-free seawater. 3. Calcium antagonist drugs were more inhibitory on ACh-induced responses than on K-responses suggesting a dichotomy of calcium channel activities modulated by these agonists. 4. The calcium ionophore A23187 enhanced ACh-induced responses of both muscles but was without effect on K-induced responses. 5. The responses of these Neptunea muscles to calcium antagonist drugs show some similarities but also differences to those of Buccinum muscles and are quite unlike the excitation induced by organic antagonists in similar muscles of the American whelk Busycon canaliculatum.

Pharmacology of the serotonin-induced meiosis reinitiation in Spisula Solidissima oocytes

Germinal vesicle breakdown (GVBD) is the first visible response of the oocyte of Spisula solidissima to the neurohormone serotonin. Pharmacological characterization of this response was performed by using 24 serotonin-related compounds. Dose-response curves were assessed by quantification of GVBD. Rank orders of potency obtained were among agonists: serotonin > 8-hydroxy-2-(di- N-propylamino)tetralin hydrobromide > 2-methyl-serotonin > 1-(3-trifluoromethylphenyl)piperazine; among antagonists; ritanserin > ICS205930 > mianserin = ketanserin = propranolol > metoclopramide = yohimbine > spiperone. Various other monoaminergic compounds tested were inefficient, demonstrating the specificity of the oocyte response to serotonin. Transduction mechanisms underlying this response were then investigated. Ca 2+ appeared to be involved since serotonin induced an increase in the uptake of 45Ca 2+ and since it was inefficient in calcium-free sea water. The absence of synergy between serotonin and KCl suggested that both compounds use a common transduction pathway. Exposure of the oocyte to the protein kinase C activator TPA inhibited serotonin-dependent maturation. Our data thus point to an original, previously uncharacterized pharmacological profile and transduction mechanism by which serotonin induces oocyte meiosis reinitiation in Spisula solidissima

The contractility of elongated microvilli in early sea urchin embryos.

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