cyclic-gmp and speract

Zinc (Zn(2+)) has been recently recognized as a crucial element for male gamete function in many species although its detailed mechanism of action is poorly understood. In sea urchin spermatozoa, Zn(2+) was reported as an essential trace ion for efficient sperm motility initiation and the acrosome reaction by modulating intracellular pH (pHi). In this study we found that submicromolar concentrations of free Zn(2+) change membrane potential (Em) and increase the concentration of intracellular Ca(2+) ([Ca(2+)]i) and cAMP in Lytechinus pictus sperm. Our results indicate that the Zn(2+) response in sperm of this species mainly involves an Em hyperpolarization caused by K(+) channel activation. The pharmacological profile of the Zn(2+)-induced hyperpolarization indicates that the cGMP-gated K(+) selective channel (tetraKCNG/CNGK), which is crucial for speract signaling, is likely a main target for Zn(2+). Considering that Zn(2+) also induces [Ca(2+)]i fluctuations, our observations suggest that Zn(2+) activates the signaling cascade of speract, except for an increase in cGMP, and facilitates sperm motility initiation upon spawning. These findings provide new insights about the role of Zn(2+) in male gamete function.

Topics: Acrosome Reaction; Animals; Calcium; Cyclic GMP; Hydrogen-Ion Concentration; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; Lytechinus; Male; Membrane Potentials; Oligopeptides; Potassium Channel Blockers; Potassium Channels; Sperm Motility; Spermatozoa; Zinc

Topics: Acrosome Reaction; Animals; Calcium Signaling; Cyclic AMP; Cyclic GMP; Female; Hydrogen-Ion Concentration; Ion Transport; Male; Membrane Potentials; Oligopeptides; Potassium; Receptors, Cell Surface; Sea Urchins; Sodium; Sperm Tail; Sperm-Ovum Interactions; Spermatozoa

Speract, a sperm-activating peptide (SAP) from sea urchin eggs, increases the intracellular concentration of Ca(2+) ([Ca(2+)]i) and modulates sperm motility. We measured the initial sperm response to speract using its caged analog and observed, for the first time, a small but significant decrease in sperm [Ca(2+)]i before the increase. Both directions of the [Ca(2+)]i change were completely blocked in high K(+) seawater. Using membrane-permeant caged cyclic nucleotides (cNMP), only cGMP induced the decrease in [Ca(2+)]i although both cGMP and cAMP increased the [Ca(2+)]i. The decrease in the [Ca(2+)]i induced by cGMP was more notable following a second photolytic event, once [Ca(2+)]i had been elevated by an initial flash. This pattern of [Ca(2+)]i change was confirmed in individual sperm. These results together with pharmacological evidence suggest that the initial [Ca(2+)]i decrease is due to a Na(+)/Ca(2+) exchanger activity, stimulated by hyperpolarization mediated by K(+) efflux through cGMP-regulated K(+) channels.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Calcium; Calcium Signaling; Cell Polarity; Cyclic AMP; Cyclic GMP; Hydrogen-Ion Concentration; Kinetics; Male; Nucleotides, Cyclic; Oligopeptides; Photolysis; Sea Urchins; Sodium-Calcium Exchanger; Spermatozoa; Thiourea; Ultraviolet Rays

Sea urchin sperm motility is modulated by sperm-activating peptides. One such peptide, speract, induces changes in intracellular free calcium concentration ([Ca2+]i). High resolution imaging of single sperm reveals that speract-induced changes in [Ca2+]i have a complex spatiotemporal structure. [Ca2+]i increases arise in the tail as periodic oscillations; [Ca2+]i increases in the sperm head lag those in the tail and appear to result from the summation of the tail signal transduction events. The period depends on speract concentration. Infrequent spontaneous [Ca2+]i transients were also seen in the tail of unstimulated sperm, again with the head lagging the tail. Speract-induced fluctuations were sensitive to membrane potential and calcium channel blockers, and were potentiated by niflumic acid, an anion channel blocker. 3-isobutyl-1-methylxanthine, which potentiates the cGMP/cAMP-signaling pathways, abolished the [Ca2+]i fluctuations in the tail, leading to a very delayed and sustained [Ca2+]i increase in the head. These data point to a model in which a messenger generated periodically in the tail diffuses to the head. Sperm are highly polarized cells. Our results indicate that a clear understanding of the link between [Ca2+]i and sperm motility will only be gained by analysis of [Ca2+]i signals at the level of the single sperm.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Anion Transport Proteins; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Signaling; Cyclic AMP; Cyclic GMP; Extracellular Space; Intracellular Fluid; Male; Membrane Potentials; Nickel; Niflumic Acid; Oligopeptides; Potassium; Sea Urchins; Signal Transduction; Sperm Head; Sperm Motility; Sperm Tail

Speract, a decapeptide from Strongylocentrotus purpuratus sea urchin eggs, transiently stimulates a membrane guanylyl cyclase and activates a K(+)-selective channel that hyperpolarizes sperm. However, previous studies of sperm and of sperm membrane vesicles reached conflicting conclusions about the mechanisms that open these channels. We find that speract hyperpolarizes and increases the cGMP content of flagellar vesicles. We confirm previous findings that intravesicular GTPgammaS and GTP enhance this hyperpolarization, but not GDPbetaS. The G protein activators AlF(-)(4) and mastoparan also are ineffective. Thus, it is unlikely that a G protein participates in the speract response. In contrast, hyperpolarization responses to speract are increased by 3-isobutyl-1-methylxanthine, which preferentially inhibits cGMP-selective phosphodiesterases of sperm, and the 8Br-cGMP derivative hyperpolarizes vesicles in the absence of speract. The responses to speract and to 8Br-cGMP have similar ionic selectivities (K(+) > Rb(+) > > Li(+) > Na(+)) and sensitivities to the channel blockers 4-aminopiridine and 3, 4-dichlorobenzamil, indicating that they likely result from opening of the same K(+) channel. Inhibitors that preferentially inhibit cAMP-selective phosphodiesterases do not alter responses to speract, and permeant cAMP analogs do not hyperpolarize vesicles. In addition, inhibitors of protein kinases and phosphatases fail to alter vesicle hyperpolarization by speract. The increase in vesicular cGMP content produced by speract therefore may directly mediate opening of the channel that hyperpolarizes sperm membrane vesicles. Similar mechanisms presumably operate in intact sperm.

Topics: 4-Aminopyridine; Amiloride; Animals; Cations, Monovalent; Cell Membrane; Cyclic GMP; Enzyme Inhibitors; GTP-Binding Proteins; Guanosine Diphosphate; Male; Membrane Potentials; Oligopeptides; Phosphoprotein Phosphatases; Potassium Channel Blockers; Potassium Channels; Protein Kinase Inhibitors; Sea Urchins; Signal Transduction; Sperm Tail; Spermatozoa; Thionucleotides

The egg peptide speract stimulates sperm guanylyl cyclase and presumably enhances fertilization, but the roles of cGMP in sperm responses are yet undetermined. Here we show that speract-induced accumulation of cGMP or cAMP is selectively enhanced by the phosphodiesterase inhibitors, 3-isobutyl-1-methylxanthine (IBMX) or papaverine, respectively. These inhibitors provided the unusual opportunity to examine the consequences of manipulating cGMP- and cAMP-dependent responses. The following observations suggest that cGMP mediates activation of K channels, the earliest known ionic event in speract signal transduction: 1) both cGMP content and K+ permeability are maximal within 15 s of speract stimulation and both decline after intracellular pH (pHi) increases in response to hyperpolarization; 2) IBMX prolongs elevation of cGMP and sustains K+ permeability after pHi increases; 3) both cGMP accumulation and K+ permeability also are enhanced when the pHi increase is prevented by an elevated concentration of external K+ (Ko); 4) elevating pHi with NH4Cl bypasses the blockade imposed by high Ko and decreases K+ permeability. Because IBMX antagonizes this action of NH4Cl, these results further suggest that elevation of pHi initiates an inactivation of guanylyl cyclase that leads to K channel closure. However, K+ permeability is restored upon subsequent elevation of intracellular [Ca2+] (Cai), indicating either that sperm K channels possess an alternate regulatory mode, or that a distinct Ca(2+)-activated K permeability also participates in speract signal transduction. Regardless of the mechanism that mediates Cai action, sperm K channels are identified as downstream targets of cGMP and are implicated in a feedback loop that both terminates guanylyl cyclase activity and leads to their own inactivation.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Ammonium Chloride; Animals; Cyclic AMP; Cyclic GMP; Dimethyl Sulfoxide; Hydrogen-Ion Concentration; Kinetics; Male; Membrane Potentials; Oligopeptides; Papaverine; Potassium; Potassium Channels; Sea Urchins; Semen; Spermatozoa; Tetraethylammonium; Tetraethylammonium Compounds

Resact, a peptide obtained from eggs, causes a change in the Mr, and a loss of 32P from a plasma membrane protein identified as guanylate cyclase. Here, a resact analog (125I-[Tyr1, Ser8] resact) was synthesized and shown to bind to isolated sperm membranes. Resact, but not speract, competed with the radiolabeled ligand for binding. When membranes were prepared under appropriate conditions, guanylate cyclase remained at Mr 160,000; the incubation of membranes with gamma-32P-ATP resulted in the formation of 32P-labeled guanylate cyclase. The addition of resact to the membranes caused a shift in the Mr, a complete loss of 32P, and a 70% reduction in guanylate cyclase activity within 1 min; resact had an ED 50 at 100 nM concentration. Speract failed to cause any of these effects. This represents the first demonstration of receptor-mediated responses of isolated sperm membranes identical to those seen in the intact cell.

Topics: Animals; Binding, Competitive; Cell Membrane; Chemotaxis; Cyclic GMP; Egg Proteins; Guanylate Cyclase; Male; Membrane Proteins; Molecular Weight; Oligopeptides; Peptides; Phosphorylation; Receptors, Cell Surface; Sea Urchins; Spermatozoa

Two peptides, speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), which activate sperm respiration and motility and elevate cyclic GMP concentrations in a species-specific manner, were tested for effects on guanylate cyclase activity. The guanylate cyclase of sea urchin spermatozoa is a glycoprotein and it is localized entirely on the plasma membrane. When intact sea urchin sperm cells were incubated with the appropriate peptide for time periods as short as 5 s and subsequently homogenized in detergent, guanylate cyclase activity was found to be as low as 10% of the activity of cells not treated with peptide. The peptides showed complete species specificity and analogues of one peptide (speract) caused decreases in enzyme activity coincident with their receptor binding properties. The peptides did not inhibit enzyme activity when added after detergent solubilization of the enzyme. When detergent-solubilized spermatozoa were incubated at 22 degrees C, guanylate cyclase activity declined in previously nontreated cells to the peptide-treated level. The rate of decline was dependent on temperature and protein concentration. When spermatozoa were first incubated with 32P, the decrease in guanylate cyclase activity was accompanied by a shift in the apparent molecular weight of a major plasma membrane protein (160,000-150,000) and a loss of 32P label from the 160,000 band. Other agents (Monensin A, NH4Cl) which were capable of stimulating sperm respiration and motility also caused decreases of guanylate cyclase activity when added to intact but not detergent-solubilized spermatozoa. The maximal decrease in guanylate cyclase activity occurred 5-10 min after addition of these agents. The enzyme response to Monensin A required extracellular Na+ suggestive that the ionophore caused the effect on guanylate cyclase activity by virtue of its ability to catalyze Na+/H+ exchange. These studies demonstrate that guanylate cyclase activity of sperm cells can be altered by the specific interaction of egg-associated peptides with their plasma membrane receptors.

Topics: Ammonium Chloride; Animals; Cell Membrane; Cyclic GMP; Guanylate Cyclase; Hydrogen-Ion Concentration; Male; Molecular Weight; Monensin; Oligopeptides; Peptides; Polyethylene Glycols; Receptors, Cell Surface; Sea Urchins; Sodium; Spermatozoa; Time Factors

A low molecular weight peptide (speract) associated with sea urchin eggs has been purified to apparent homogeneity by charcoal adsorption, DEAE-Sephacel chromatography, Bio-Gel P-2 filtration, and Dowex AG 50W-X4 chromatography. Gametes from 5000 female sea urchins were required for the isolation of approximately 9 mg of the peptide. The isolated peptide is homogenous based on [3H]acetic anhydride labeling, gel filtration, and reverse phase high pressure liquid chromatography. Speract is composed entirely of neutral and acidic amino acids with glycine as the major component, and it appears to have a blocked NH2 terminus based on its insensitivity to leucine aminopeptidase, its failure to react with dansyl chloride, and its chromatographic behavior on strong cation exchange resins. Speract is a potent stimulator of sea urchin sperm oxygen consumption, causing significant increases of sperm respiration rates at concentrations as low as 10(-12) M and producing 20-fold increases of oxygen consumption at maximal concentrations of 10(-8) M. Sperm cyclic GMP and cyclic AMP concentrations are also increased by speract, but concentrations of at least 10(-10) M and 10(-9) M are required for half-maximal elevations, respectively. The peptide, purified from Strongylocentrotus purpuratus eggs, also cross-reacts with spermatozoa from Lytechnis pictus sea urchins, suggesting that speract does not show species specificity. These results represent the first report of the purification of a peptide associated with eggs that may affect spermatozoa under natural conditions.

Topics: Amino Acids; Animals; Biological Assay; Cyclic AMP; Cyclic GMP; Endopeptidases; Female; Male; Oligopeptides; Ovum; Oxygen Consumption; Sea Urchins; Spermatozoa

Topics: Animals; Biological Transport, Active; Cyclic AMP; Cyclic GMP; Furans; Hydrogen-Ion Concentration; Male; Monensin; Oligopeptides; Oxygen Consumption; Sea Urchins; Sodium; Sperm Motility; Spermatozoa