Free Seawater Research Articles

Pulsatile urea excretion in gulf toadfish (Opsanus beta): evidence for activation of a specific facilitated diffusion transport system.

When toadfish are made ureotelic by a crowding/confinement protocol, they excrete approximately 90 % of their urea nitrogen (urea-N) production in large, irregular pulses (1-2 pulses per day) from the gill region. We investigated three hypotheses as to the mechanism of pulsatile excretion: (i) the presence of an active reabsorptive 'back-transport' mechanism that is periodically inhibited to allow urea-N excretion to occur; (ii) the periodic occurrence of a generalized, non-specific increase in gill permeability; and (iii) the presence of a specific facilitated diffusion transport system that is periodically activated. Exposure of toadfish during non-pulse periods to treatments designed to block a 'back-transport' mechanism (Na+-free sea water or the urea analogues 30 mmol l-1 thiourea or 30 mmol l-1 acetamide in the external water) did not stimulate a leakage of urea-N, thereby opposing the first hypothesis. The second hypothesis was opposed by several results. Neither injection of the potent branchial vasodilator L-isoprenaline (10(-5) mol l-1) nor infusion of NH4Cl, the latter at levels known to stimulate urea-N efflux in perfused gills, had any effect on urea-N excretion. Furthermore, during natural pulse events, when the normally very low gill permeability to urea (3x10(-7) cm s-1) increased at least 35-fold, there was no accompanying increase in permeability to either 3H2O (1.5x10(-5) cm s-1) or the paracellular marker [14C]PEG-4000 (10(-8) cm s-1). However [14C]thiourea permeability (1.5x10(-7) cm s-1) increased approximately fivefold, in support of the third hypothesis. Furthermore, when 30 mmol l-1 urea was placed in the external water, a concentration (60 000 micromol-N l-1) approximately three times that of blood (20 000 micromol-N l-1), each efflux pulse event (measured with [14C]urea) was accompanied by a net uptake, such that blood urea-N levels rose rather than fell. A proportional 1:1 relationship between influx per unit external concentration and efflux per unit internal (i.e. plasma) concentration indicated a fully bidirectional transport system. The simultaneous presence of 60 mmol l-1 thiourea in the external water inhibited the influx component by 73 %, further supporting this conclusion. These data, together with recent molecular, morphological and endocrinological evidence, strongly suggest that pulsatile urea-N excretion is caused by the periodic activation of a facilitated urea transporter in the gills, similar to the vasopressin-regulated urea transporter in the mammalian kidney.

Repair of hair bundles in sea anemones by secreted proteins

Sea anemones are sessile invertebrates that detect movements of prey using numerous hair bundles located on tentacles surrounding their mouth. Previously we found that hair bundles of anemones are structurally and functionally similar to those of vertebrates. After 10–15 min exposure to calcium depleted buffers, hair bundles in chickens suffer moderate damage from which they recover in 12 h without requiring new protein synthesis [Zhao, Yamoah and Gillespie, Proc. Natl. Acad. Sci. USA 94 (1996) 15469–15474]. We find that after 1 h exposure to calcium free seawater, hair bundles of anemones suffer extensive damage from which they recover in 4 h, apparently because of newly synthesized, secretory proteins called `repair proteins'. Recovery is delayed in a dose dependent fashion by cycloheximide. In the presence of exogenously added repair proteins, recovery occurs within 8 min and is cycloheximide insensitive. Recovery is ascertained by a bioassay performed on intact specimens, by electrophysiology, and by timelapse video microscopy. Fraction β, a chromatographic fraction with bioactivity comparable to the complete mixture of repair proteins, consists of complexes having an estimated mass of 2000 kDa. Avidin based cytochemistry suggests that biotinylated fraction β binds to damaged hair bundles. SDS-PAGE gel electrophoresis demonstrates that fraction β contains 8–10 polypeptides of 90 kDa or smaller. At least four of these polypeptides apparently are consumed during the repair process. Negatively stained samples of fraction β are shown by transmission electron microscopy to include filamentous structures similar in length (150 nm) and width (6 nm) to linkages between stereocilia. The filamentous structures can be associated with globular structures (20 nm in diameter). A model is presented wherein repair proteins comprise replacement linkages and enzymes that attach linkages to appropriate membrane proteins.

Large plasma membrane disruptions are rapidly resealed by Ca2+-dependent vesicle-vesicle fusion events.

A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390-393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an approximately 40 by 10 microm surface patch was torn off, entry of fluorescein stachyose (FS; 1, 000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (approximately 5-10 s) rise in cytosolic Ca2+ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was approximately 3 mM Ca2+ (SW is approximately 10 mM Ca2+). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca2+ -free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet-rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca2+ regulated vesicle-vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion reaction is to form a replacement bilayer patch. This patch is added to the discontinuous surface bilayer by exocytotic fusion events.

Ascidian eggs block polyspermy by two independent mechanisms: One at the egg plasma membrane, the other involving the follicle cells

Many ascidians live in clumps and usually release sperm before the eggs. Consequently, eggs are often spawned into dense clouds of sperm. Because fertilization by more than a single sperm is lethal, ascidians have evolved at least two successive blocks to polyspermy: the rapid release of a glycosidase that inhibits sperm binding to the vitelline coat (VC) and a subsequent change in membrane potential that prevents supernumerary sperm–egg fusion. This paper shows that (1) these two blocks can be uncoupled by the use of suramin, and (2) most of the glycosidase appears to be from the follicle cells, which are accessory cells on the outside of the egg VC. Phallusia mammillata eggs initially bind numerous sperm but, after the glycosidase is released, only a few additional sperm bind. Intact eggs in 20 μM suramin release glycosidase, but the electrical response is inhibited; sperm swim actively and bind to the VC but fail to penetrate. Suramin treatment is completely reversible; intact eggs exhibit the electrical response an average of 11 minutes after the drug is washed out. Sperm must contact the follicle cells before passing through the VC; eggs with the VC removed and fertilized in the presence of 20 μM suramin show the electrical response 35% of the time, thus VC removal enhances sperm entry. Like the intact eggs, 100% of the naked eggs respond electrically to fertilization after the drug is washed out. Follicle cells that are isolated by calcium magnesium free seawater and then returned to complete seawater release N-acetylglucosaminidase activity in response to sperm. Thus, these eggs have two blocks to polyspermy that operate in sequence: an early first block resulting from enzymatic modification of the VC by N-acetylglucosaminidase released primarily from follicle cells and a second electrical block operating at the egg plasma membrane level and requiring sperm–egg fusion. Mol. Reprod. Dev. 48:137-143, 1997. © 1997 Wiley-Liss, Inc.

Ascidian eggs block polyspermy by two independent mechanisms: one at the egg plasma membrane, the other involving the follicle cells.

Many ascidians live in clumps and usually release sperm before the eggs. Consequently, eggs are often spawned into dense clouds of sperm. Because fertilization by more than a single sperm is lethal, ascidians have evolved at least two successive blocks to polyspermy: the rapid release of a glycosidase that inhibits sperm binding to the vitelline coat (VC) and a subsequent change in membrane potential that prevents supernumerary sperm-egg fusion. This paper shows that (1) these two blocks can be uncoupled by the use of suramin, and (2) most of the glycosidase appears to be from the follicle cells, which are accessory cells on the outside of the egg VC. Phallusia mammillata eggs initially bind numerous sperm but, after the glycosidase is released, only a few additional sperm bind. Intact eggs in 20 microM suramin release glycosidase, but the electrical response is inhibited; sperm swim actively and bind to the VC but fail to penetrate. Suramin treatment is completely reversible; intact eggs exhibit the electrical response an average of 11 minutes after the drug is washed out. Sperm must contact the follicle cells before passing through the VC; eggs with the VC removed and fertilized in the presence of 20 microM suramin show the electrical response 35% of the time, thus VC removal enhances sperm entry. Like the intact eggs, 100% of the naked eggs respond electrically to fertilization after the drug is washed out. Follicle cells that are isolated by calcium magnesium free seawater and then returned to complete seawater release N-acetylglucosaminidase activity in response to sperm. Thus, these eggs have two blocks to polyspermy that operate in sequence: an early first block resulting from enzymatic modification of the VC by N-acetylglucosaminidase released primarily from follicle cells and a second electrical block operating at the egg plasma membrane level and requiring sperm-egg fusion.

Hair bundles of sea anemones as a model system for vertebrate hair bundles

Sea anemones are marine invertebrates that use hair bundles to detect swimming movements of prey. Prey are captured by nematocysts (stinging capsules) that discharge into the prey. To further characterize anemone hair bundles and to compare hair bundles in anemones with hair bundles in vertebrates, we investigated fine structure and cytochemistry of anemone hair bundles. In addition, using a biological assay based on counting nematocysts discharged into vibrating test probes, we examined sensitivity of vibration detection to aminoglycoside antibiotics, Ca2+-free seawater, and amiloride. Like vertebrate hair bundles, anemone hair bundles are composed of stereocilia, possess lateral linkages between stereocilia whose preservation for transmission electron microscopy is enhanced by ruthenium red, and possess tip links morphologically similar to vertebrate tip links. Furthermore, vibration-dependent discharge of nematocysts is reversibly inhibited by 10−4 M streptomycin and abolished by brief exposure to Ca2+-free seawater. However, unlike vertebrate hair bundles, anemone hair bundles appear to be insensitive to amiloride since vibration-dependent discharge of nematocysts is unaffected by up to mM amiloride. Thus, anemone hair bundles may serve as a useful model system for vertebrate hair bundles with the interesting feature of being insensitive to amiloride.

26] Bioassay for determining endogenous levels of cyclic ADP-ribose

Publisher Summary This chapter describes a bioassay for cyclic ADP-ribose (ADPR) on the basis of its Ca 2+ -releasing activity in homogenates prepared from sea urchin eggs. Substances that could potentially interfere with the Ca 2+ release bioassay, such as Ca 2+ , inositol 1,4,5-triphosphate, and NAD + , must be removed from the sample. Cyclic ADP-ribose in the extracts is purified by a two-step system consisting of phenylboronate chromatography followed by anion-exchange chromatography on AG MP-1. Strongylocentrotus purpuratus eggs are obtained by stimulating ovulation of a female sea urchin with a 1-ml injection of 0.5 M KCI and washed once in artificial seawater, twice in Ca 2+ -free seawater containing 1 m M ethylene glycol tetraacetic acid (EGTA), twice in Ca 2+ -free seawater without EGTA, once with the homogenization buffer, and resuspended with the same medium to 25%. The sensitivity of the bioassay can be increased by pretreating the homogenate with caffeine, which is known to potentiate cADPR-induced Ca 2+ release.

Parthenogenetic activation of mouse oocytes by 6-dimethylaminopurine

We have investigated the increase of phosphorylated proteins upon activation of surf clam (Spisula solidissima) oocytes, by measuring the cumulative incorporation of 32P in proteins and by performing an SDS-PAGE and autoradiographic analysis of 32P-labeled proteins, from oocytes initially radiolabeled with 32P-orthophosphate. The phosphorylation inhibitor 6-dimethylaminopurine (6-DMAP) inhibits both germinal vesicle breakdown (GVBD) and the normal increase in phosphorylated proteins observed upon activation by KCl, in a reversible and dose-dependent manner. Using different artificial seawaters (normal, Ca2+-free, Na+-free), we observed that the increase of phosphorylated proteins, upon K+ stimulation, occurs only when GVBD is allowed to proceed along with an increased Ca2+ influx, in normal or Na+-free seawater. Stimulation of oocytes by ammonia, which directly raises intracellular pH (pHi) but does not trigger GVBD, is without effect on the level or pattern of phosphorylated proteins. The link between the Ca2+ influx and the level of phosphorylated proteins was further investigated using conditions altering the duration or the level of Ca2+ influx upon K+ stimulation. In all conditions tested, both GVBD and the level of phosphorylated proteins were similarly affected by alterations of the Ca2+ influx, indicating that these processes are tightly coupled one with another. Upon activation of oocytes, six major proteins of estimated molecular weights of 31, 41, 48, 56, 80 and 86 kDa undergo an increased phosphorylation that is reversibly sensitive to 6-DMAP. Our results suggest that increased protein phosphorylation, sensitive to 6-DMAP, is necessary for GVBD and that it is indirectly linked to the increased Ca2+ influx that stands as an upstream trigger for activation, while an elevated pHi alone has no effect on these processes.

Deep-ocean field test of methane hydrate formation from a remotely operated vehicle

We have observed the process of formation of clathrate hydrates of methane in experiments conducted on the remotely operated vehicle (ROV) Ventana in the deep waters of Monterey Bay. A tank of methane gas, acrylic tubes containing seawater, and seawater plus various types of sediment were carried down on Ventana to a depth of 910 m where methane gas was injected at the base of the acrylic tubes by bubble stream. Prior calculations had shown that the local hydrographic conditions gave an upper limit of 525 m for the P-T boundary defining methane hydrate formation or dissociation at this site, and thus our experiment took place well within the stability range for this reaction to occur. Hydrate formation in free seawater occurred within minutes as a buoyant mass of translucent hydrate formed at the gas-water interface. In a coarse sand matrix the filling of the pore spaces with hydrate turned the sand column into a solidified block, which gas pressure soon lifted and ruptured. In a fine-grained black mud the gas flow carved out flow channels, the walls of which became coated and then filled with hydrate in larger discrete masses. Our experiment shows that hydrate formation is rapid in natural seawater, that sediment type strongly influences the patterns of hydrate formation, and that the use of ROV technologies permits the synthesis of large amounts of hydrate material in natural systems under a variety of conditions so that fundamental research on the stability and growth of these substances is possible.

Calcium and the photopolarization ofPelvetia zygotes

The initially apolar zygotes of the brown algae,Fucus andPelvetia, form their main axes during the hours following fertilization and each cell expresses its axis by germinating at one location. The germinating region is destined to become the rhizoid and the rest of the zygote gives rise to the thallus. In response to unilateral blue light, the zygotes organize their developmental axes so that the rhizoids emerge on the shaded side, away from the light source. In the research reported here, the signaltransduction elements involved in the photopolarization ofPelvetia fastigiata De Toni zygotes have been investigated. It was found that exposure of zygotes to 90or 150-min pulses of unilateral light in the absence of extracellular Ca2+ completely eliminated photopolarization; that is, the cells formed their rhizoid-thallus axes randomly with respect to the light direction, while controls similarly exposed to light in normal (10 mM) Ca2+ were well polarized. When the cells were incubated in Ca2+-free sea water for an hour before being given the light pulse (while still in Ca2+-free sea water), they exhibited an unusual negative polarization: they formed their rhizoids on the hemisphere nearer the light source. Organic and inorganic calcium-channel blockers reduced or abolished photopolarization when present during light pulses. Reducing external Ca2+ to one-tenth of normal has the paradoxical effect of increasing calcium influx intoPelvetia zygotes. When zygotes were given light pulses in reduced extracellular calcium, the degree of photopolarization was increased substantially. These data are consistent with the idea that the formation of an intracellular gradient of [Ca2+] is an essential part of the polarization process. The fungus-derived calmodulin antagonist, ophiobolin A, blocked or greatly delayed germination when present continuously at a concentration of 100–300 nM. However, when present at 300 nM during a brief light pulse, it markedly increased the sensitivity of the cells to light. These results suggest that calmodulin may be the mediator of intracellular [Ca2+] gradients in the photopolarization process.

A Simple Method for Introducing Macromolecules into the Blastocoel of Living Starfish Embryos

Abstract We have devised a simple method for introducing macromolecules into the blastocoel of living starfish embryos. In this method, embryos are treated for 15 min with Ca2+ free sea water (CFSW) containing 0.1–5.0 mg/ml of enzymes or immunoglobulins. These molecules are considered to flow through the septate junction, the septa of which are made diffuse by deprivation of Ca2+ ions from the environmental sea water. Introduction of enzymes which affect the component molecules of the extracellular matrix (ECM) drastically deformed the larval morphology. Introduction of a monoclonal antibody recognizing an ECM component prevented the mesenchyme cells from stretching and migrating normally. These results show that our method can be used to study the function of various ECM components in embryonic development.

Microfilament Contraction Promotes Rounding of Tunic Slices: An Integumentary Defense System in the Colonial Ascidian Aplidium yamazii.

In Aplidium yamazii, when a slice of a live colony (approximately 0.5 mm thick) was incubated in seawater for 12 h, the slice became a round tunic fragment. This tunic rounding was inhibited by freezing of the slices, incubation with Ca2+-Mg2+ -free seawater, or addition of cytochalasin B. Staining of microfilaments in the slices with phalloidin-FITC showed the existence of a cellular network in the tunic. Contraction of this cellular network probably promotes rounding of the tunic slice. In electron microscopic observations, a new tunic cuticle regenerated at the surface of the round tunic fragments; the tunic cuticle did not regenerate in newly sliced specimens nor in specimens in which rounding was experimentally inhibited. Based on these results, an integumentary defense system is proposed in this species as follows. (1) When the colony is wounded externally, contraction of the cellular network promotes tunic contraction around the wound. (2) The wound is almost closed by tunic contraction. (3) Tunic contraction increases the density of the filamentous components of the tunic at the wound, and it may accelerate the regeneration of tunic cuticle there.

Effect of Ca2+ -free Seawater Treatment on 1-Methyladenine Production in Starfish Ovarian Follicle Cells: (starfish follicle cells/1-MeAde/GSS/cAMP/Ca2 +-free seawater).

When follicle cells from ovaries of the starfish Asterina pectinifera were washed with Ca2+ -free seawater (CaFSW), the production of 1-methyladenine (1-MeAde) in response to gonad-stimulating substance (GSS) decreased to a large degree. The cyclic AMP content of CaFSW-treated follicle cells was much lower than that of non-treated cells, and the level was increased slightly by GSS, but not to a degree sufficient for production of 1-MeAde. The production of 1-MeAde and cyclic AMP in the presence of GSS was dependent on extracellular Ca2+ concentration, and was considerably reduced at a Ca2+ concentration below 1 mM. Thus, the decrease of 1-MeAde production by follicle cells after treatment with CaFSW is due to the low levels of cAMP. Furthermore, an ADP-ribosylation experiment using [α-32 P]NAD+ in the presence of cholera toxin and pertussis toxin with membrane preparations of follicle cells treated with CaFSW revealed the presence of two types (stimulatory and inhibitory) of G-proteins. However, activity of the adenylyl cyclase was not influenced by GSS regardless of the presence or absence of GTP. These findings may suggest that GSS is unable to bind to its receptor in follicle cells after washing with CaFSW.

Onset of DNA synthesis in experimentally activated ascidian eggs

AbstractDNA synthesis was studied autoradiographically in unfertilized ascidian eggs (Ascidia malaca and Ciona intestinalis) that had been artificially activated by a K+‐free external medium or a Ca‐ionophore. Naked eggs of A. malaca were incubated in K+‐free seawater that contained [3H]‐thymidine for 30 min, and naked eggs of C. intestinalis were incubated in seawater supplemented with a Ca‐ionophore and [3H]‐thymidine for 15 min. The observations revealed limited activation in eggs treated with K+‐free seawater. Activation consisted of surface modifications and the onset of DNA synthesis. Incorporation of [3H]‐thymidine was detected in female nuclei of treated eggs which had not ejected their polar bodies. In eggs treated with the Ca‐ionophore, a known activating agent, the rate of incorporation of [3H]‐thymidine was higher and nuclear duplication was also observed. Comparison of activation under the two different sets of artificial conditions indicated that, in K+‐free medium, the early calcium‐dependent events, such as deformation of egg shape, extrusion of the polar bodies, and ooplasmic segregation, do not occur, while DNA synthesis is initiated. The results are discussed on the basis of the hypothesis that, in Ascidians, egg‐activation events are only partly regulated by an intracellular increase in levels of calcium ions. DNA synthesis appears to be an independent process that is initiated by changes in the egg plasma membrane. © 1994 Wiley‐Liss, Inc.

A Protein That Binds an Exogastrula-Inducing Peptide, EGIP-D, in the Hyaline Layer of Sea Urchin Embryos: (exogastrula-inducing peptide (EGIP)/binding protein/hyaline layer/sea urchin/exogastrulation).

Exogastrula-inducing peptides are present in eggs and embryos of the sea urchin Anthocidaris crassispina. They induce exogastrulation when added exogenously to the embryos. In the present study, we investigated an EGIP-D-binding protein in the embryos. EGIP-D was incubated with homogenates of embryos. EGIP-D was then cross-linked to the binding protein by use of disuccinimidyl suberate (DSS) and the complex was analyzed by western blotting with an EGIP-D-specific antibody. A 30-kDa protein was detected in both eggs and embryos. To examine the localization of this protein, EGIP-D was added to intact embryos, cross-linked to proteins by use of DSS, and the complexes were again analyzed by western blotting. The EGIP-D-binding protein was detected in intact embryos but not in embryos treated with Ca2+ - and Mg2+ -free seawater (CMF-SW) that removes the hyaline layer (HL). It appeared, therefore, that this protein was present on the outer surface of the embryo, being a constituent of the HL. The CMF-SW extract that contained EGIP-D-binding protein, inhibited the induction of exogastrulation by EGIP-D. Furthermore, the treatment of embryos with CMF-SW prevented EGIP-D from inducing exogastrulation. Our observations indicate that the interaction between EGIP-D and the binding protein is a prerequisite for induction of exogastrulation by EGIP-D.

Sperm-egg fusion in the sea urchin is blocked in Mg2+-free seawater

Magnesium ions as well as calcium ions are required for successful fertilisation in sea urchins. In the absence of Mg2+ spermatozoa attached to the egg plasma membrane, their acrosomal processes passing through the vitelline envelope, but could not enter the egg cytoplasm (Sano et al., Dev. Growth Differ. 22, 531-41, 1980). Such an individual spermatozoon was observed microscopically to resume entry into the egg immediately after the addition of a sufficient amount of Mg2+ to the surrounding medium. Neither any change in membrane potential nor an increase in intracellular Ca2+ concentration of the egg was observed after insemination in the absence of Mg2+, although both could be observed after the addition of Mg2+. The sperm heads did not show fluorescence when attached to the surface of an egg previously microinjected with mithramycin A in Mg-free seawater, indicating that there was no connection between the sperm and the egg. Therefore, occurrence of fertilisation potential must be a post-fusional event. These results suggest that Mg2+ are indispensable for fusion between the sperm acrosomal membrane and the egg plasma membrane.

Ionic factors affecting motility, respiration and fertilization rate of the sperm of the bivalvePecten maximus (L.)

Fertilization of the scallopPecten maximus occurs after gametes were naturally released in sea water by the bivalve which has undergone stimulation. The motility of the spermatozoa requires their dilution in sea water (1/40). Dilution triggers an immediate increase of oxygen consumption by sperm, reflecting an activation of a cyanide-sensitive respiration of a cellular origin. When scallops were stimulated by thermal shocks or by serotonin injection, sperm sampled at the urogenital pore output duct shows a respiration-motility activation after sea water dilution which is not seen in sperm scarified from the gonad. Dilution of kidney-sampled sperm into acidic (pH 5) or Na+-free artificial sea water reversibly inhibits both respiration and motility. In all cases fertilization rate of sperm is correlated to the increase of respiratory rate and motility measured after dilution in different media. Whether the scallop was stimulated or not, the pH of haemolymph and pericardic fluids were one pH unit below the value of sea water, the pH of the gonad and of the kidney tissues being more acidic (6.5 in average). Our results suggest that the acidic pH of the genital tract maintains the spermatozoa in a quiescent state and that capacitation occurs when male gametes move from the gonad to the kidney from where it is naturally released.

Low-Na+ Seawater Induces the Acrosome Reaction and Histone Degradation of Starfish Sperm in the Absence of Egg Jelly: (starfish/sperm/histone degradation/acrosome reaction/low-Na+ seawater).

Egg jelly induces the degradation of histones as well as the acrosome reaction in the spermatozoa of Asterina pectinifera. Much similar degradation of histones without any apparent morphological changes such as the acrosome reaction was induced in the spermatozoa by merely dispersing them into Na+ -free seawater. It required external Ca2+ much less than the jelly-induced one in normal seawater, and was not susceptible to Ca2+ -channel antagonists, verapamil and diltiazem. Once spermatozoa were incubated with egg jelly in Ca2+ -free seawater, they did not undergo the histone degradation even after subsequent addition of Ca2+ , but Na+ -free seawater rescued such blockage. Spontaneous acrosome reaction occurred in seawater containing 10-30 mM Na+ in a Ca2+ -dependent manner. This reaction was accompanied by a rapid increase in intracellular pH (pHi) followed by a large pHi decrease. Diltiazem blocked a large decrease in pHi but scarcely inhibited the acrosome reaction induced by low-Na+ seawater. Increasing K+ inhibited both pHi changes and the acrosome reaction induced by low-Na+ seawater. Decreasing pH of seawater also inhibited the pHi changes but did not affect the acrosome reaction. Strontium was also effective to induce a rapid increase, followed by a gradual decrease, in pHi and the acrosome reaction.

Effects of changes in environmental calcium on prolactin secretion in Japanese eel, Anguilla japonica

Effects of changes in environmental Ca2+ on the secretion of prolactin, a possible hypercalcemic hormone, were examined both in vivo and in vitro in the Japanese ecl, Anguilla japonica. Transfer of seawater- or freshwater-adapted fish to fresh water, fresh water containing 10 mmol Ca2+ · 1-1 sea water, Ca2+-free sea water, or deionized water was accompanied by significant changes in plasma Ca2+ levels after 7 days, except for the fish transferred from fresh water to fresh water and from sea water to sea water. Changes in external Ca2+ concentrations did not affect plasma prolactin levels, although plasma prolactin levels as well as pituitary prolactin contents were significantly greater in fish in a hypotonic environment than those in a hypertonic environment, regardless of the external Ca2+ concentration. Hypercalcemia, induced by removal of the corpuscles of Stannius, did not alter plasma prolactin levles. Incubation of the pituitary in the medium with different Ca2+ concentrations (up to 2.9 mmol·l-1) did not affect the basal release of prolactin, except at an extremely low Ca2+ concentration (less than 0.1 mmol·l-1) where prolactin release was inhibited. Addition of Ca2+ ionophore (A23187) to the medium led to a marked and significant increase in prolactin release, indicating that an increase in intracellular Ca2+ stimulates prolactin release. However, the effect was not specific to prolactin cells; a similar increase was seen in growth hormone release. These results indicate that changes in environmental Ca2+ concentration may not be the primary factor influencing prolactin secretion in the eel; changes in environmental osmolality or Na+ levels seem to be more critical for the regulation of prolactin secretion.

Role of cell interactions in ascidian muscle and pigment cell specification.

Muscle and brain pigment cell specification was studied by disrupting cell adhesion, cell dissociation, and reaggregation in embryos of the ascidianStyela clava. Treatment of embryos with Ca2+-free sea water between the 2-cell and gastrula stages disrupted blastomere adhesion but did not prevent acetylcholinesterase or muscle actin expression in presumptive muscle cells. Similar treatments initiated between the 2- and 32-cell stages caused more ectoderm cells to express tyrosinase and develop pigment granules than expected from the cell lineage. Whereas 2 pigment cells become the otolith and ocellus sensory organs in normal embryos, up to 33 pigment cells could differentiate in embryos after disruption of cell adhesion. Replacement of Ca2+-free sea water with normal sea water restored cell adhesion and usually resulted in development of embryos containing the conventional number of pigment cells. Dissociation of embryos into single cells between the 2- and 64-cell stages and culture of these cells beyond the fate restricted stage had no effect on the accumulation of muscle actin mRNA and muscle actin synthesis, but blocked pigment cell differentiation. Reaggregation of the dissociated cells did not enhance the number of cells that developed muscle features, but rescued pigment cell development. The results indicate that ascidian muscle cell specification occurs by an autonomous mechanism, whereas pigment cell specification occurs by a conditional mechanism involving cell interactions. In addition, the results suggest that negative cell interactions may restrict the potential for pigment cell development in the ectoderm of cleaving ascidian embryos.