cyclic-gmp and resact

The events that occur during chemotaxis of sperm are only partly known. As an essential step toward determining the underlying mechanism, we have recorded Ca2+ dynamics in swimming sperm of marine invertebrates. Stimulation of the sea urchin Arbacia punctulata by the chemoattractant or by intracellular cGMP evokes Ca2+ spikes in the flagellum. A Ca2+ spike elicits a turn in the trajectory followed by a period of straight swimming ('turn-and-run'). The train of Ca2+ spikes gives rise to repetitive loop-like movements. When sperm swim in a concentration gradient of the attractant, the Ca2+ spikes and the stimulus function are synchronized, suggesting that precise timing of Ca2+ spikes controls navigation. We identified the peptide asterosap as a chemotactic factor of the starfish Asterias amurensis. The Ca2+ spikes and swimming behavior of sperm from starfish and sea urchin are similar, implying that the signaling pathway of chemotaxis has been conserved for almost 500 million years.

Topics: Animals; Arbacia; Asterias; Calcium; Chemotaxis; Cyclic GMP; Flagella; Male; Peptides; Spermatozoa; Time Factors

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

Analogues of resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-LeuNH2) were synthesized to determine whether or not a stimulation of sperm respiration could be obtained independent of elevations of cyclic nucleotide concentrations. Modification of the CO2-terminal leucine NH2 did not alter biological activity; however, substitution of the two cysteinyl residues by Ser or Tyr or methylation of the cysteinyl residues resulted in divergent relative potencies dependent on whether respiration rates or cyclic nucleotide concentrations were measured. [Ser1,Tyr8]resact, as an example, was approximately 40% as potent as resact at stimulating respiration rates but was 1% as effective as resact at causing cyclic GMP elevations. An NH2-terminal fragment (Cys-Val-Thr-Gly-Ala-Pro-Gly) neither stimulated respiration nor elevated cyclic nucleotide levels at concentrations up to 10 microM whereas a CO2-terminal fragment (Cys-Val-Gly-Gly-Gly-Arg-LeuNH2) had approximately 20% of the respiration activity and 0.1% of the cyclic GMP elevating activity of resact. When the CO2- and NH2-terminal fragments were added simultaneously, however, cyclic nucleotide concentrations were elevated at the same relative concentrations as observed with resact. An analogue (125I-[Tyr1,Ser8]resact) was subsequently synthesized and used for receptor binding studies. Both the NH2-terminal and CO2-terminal fragments competed for binding, although they were 0.0004 and 0.025 times as effective as resact, respectively. However, in the presence of 1 microM resact-(1-7), resact-(8-14) was almost as potent as resact in the competitive binding assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cyclic AMP; Cyclic GMP; Kinetics; Male; Oxygen Consumption; Peptides; Sea Urchins; Spermatozoa; Structure-Activity Relationship

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 peptide (resact) associated with the eggs of the sea urchin, Arbacia punctulata, which stimulates sperm respiration rates by 5-10-fold, was purified and its amino acid sequence was determined. The sequence was found to be Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2. The peptide was subsequently synthesized by solid phase methods, amidated at the carboxyl-terminal Leu, and shown to be identical to the isolated, native material. The peptide half-maximally stimulated A. punctulata spermatozoan respiration at 0.5 nM and half-maximally elevated cyclic GMP concentrations at 25 nM at an extracellular pH of 6.6. The increase in oxygen consumption was coupled with a stimulation of motility. However, at elevated extracellular pH (pH 8.0), resact failed to appreciably stimulate respiration while the elevations of cyclic GMP continued to occur. Resact did not cross-react with sperm cells obtained from Lytechinus pictus or Strongylocentrotus purpuratus; a peptide (speract) obtained from S. purpuratus eggs (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) which activates S. purpuratus sperm respiration did not stimulate A. punctulata spermatozoa. Resact caused a shift in the apparent molecular weight (160,000-150,000) of a major sperm plasma membrane protein; as with cyclic GMP elevations, this response was evident at extracellular pH values of both 6.6 and 8.0. The protein exists in the cell as a phosphoprotein and 32P is released coincident with the molecular weight change. Approximately 115 nM resact caused one-half-maximal conversion of the 160,000-dalton protein after 1 min of incubation. Resact caused the apparent molecular weight conversion of the protein within 5 s and appeared to do so in an irreversible manner. The molecular weight change of the protein was also observed after the addition of monensin A (25 microM) and NH4Cl (40 mM), two agents known to elevate intracellular pH and to increase sperm respiration rates. The membrane protein appears to be the enzyme guanylate cyclase, but since concentrations of resact causing one-half-maximal conversion of the Mr = 160,000 form of the enzyme are about 250 times higher than those causing one-half-maximal stimulation of respiration, the relationship of the apparent molecular weight conversion to a subsequent physiological event remains unclear.

Topics: Amino Acid Sequence; Ammonium Chloride; Animals; Cell Membrane; Cyclic GMP; Electrophoresis, Polyacrylamide Gel; Female; Guanylate Cyclase; Male; Membrane Proteins; Molecular Weight; Oxygen Consumption; Peptides; Sea Urchins; Sperm-Ovum Interactions; Spermatozoa