Speract

Speract receptors are localized on sea urchin sperm flagella using a fluorescent peptide analog

In two species of sea urchins, Strongylocentrotus purpuratus and Lytechinus pictus, the egg jelly-associated decapeptide, speract, binds to specific sperm surface receptors resulting in increased sperm motility and respiration rate. Previously, a peptide analog, GGG[Y2]-speract, was used to identify a 77-kDa receptor on intact sperm cells using chemical cross-linking. In this paper we describe the synthesis and characterization of a fluorescent derivative of GGG[Y2]-speract for use as a probe for the sperm receptor. Fluorescein isothiocyanate (FITC) was conjugated to the amino terminus of GGG[Y2]-speract and the resulting analog (FITC-GGG[Y2]-speract) was purified by size exclusion chromatography and reverse-phase HPLC. Competition binding studies with the fluorescent peptide and intact spermatozoa yielded IC50 values which were indistinguishable from native speract and GGG[Y2]-speract (approximately 20 nM). FITC-GGG[Y2]-speract half-maximally stimulated sperm respiration at a concentration nearly identical to that of the native peptide (EC50 approximately 50 pM). Using digitally enhanced video imaging fluorescence microscopy, FITC-GGG[Y2]-speract was used to localize the speract receptor on the flagella of intact sperm. Excess concentrations of both unlabeled speract and GGG[Y2]-speract abolished the binding of the fluorescent analog, yet unrelated peptides did not. Further, results of cross-linking experiments using 125I-GGG[Y2]-speract and purified sperm flagella and heads were consistent with the fluorescent labeling results on whole cells. The finding that the speract receptor is localized exclusively to the sperm flagella may reveal its role in the regulation of flagellar motility.

Speract, a sea urchin egg peptide that regulates sperm motility, also stimulates sperm mitochondrial metabolism

Sea urchin sperm have only one mitochondrion, that in addition to being the main source of energy, may modulate intracellular Ca(2+) concentration ([Ca(2+)]i) to regulate their motility and possibly the acrosome reaction. Speract is a decapeptide from the outer jelly layer of the Strongylocentrotus purpuratus egg that upon binding to its receptor in the sperm, stimulates sperm motility, respiration and ion fluxes, among other physiological events. Altering the sea urchin sperm mitochondrial function with specific inhibitors of this organelle, increases [Ca(2+)]i in an external Ca(2+) concentration ([Ca(2+)]ext)-dependent manner (Ardón, et al., 2009. BBActa 1787: 15), suggesting that the mitochondrion is involved in sperm [Ca(2+)]i homeostasis. To further understand the interrelationship between the mitochondrion and the speract responses, we measured mitochondrial membrane potential (ΔΨ) and NADH levels. We found that the stimulation of sperm with speract depolarizes the mitochondrion and increases the levels of NADH. Surprisingly, these responses are independent of external Ca(2+) and are due to the increase in intracellular pH (pHi) induced by speract. Our findings indicate that speract, by regulating pHi, in addition to [Ca(2+)]i, may finely modulate mitochondrial metabolism to control motility and ensure that sperm reach the egg and fertilize it.

Speract-receptor interaction and the modulation of ion transport in Strongylocentrotus purpuratus sea urchin sperm

Speract induces calcium oscillations in the sperm tail

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.

Speract. Purification and characterization of a peptide associated with eggs that activates spermatozoa

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.