Calmodulin antagonists inhibit sea urchin sperm hyperpolarization necessary for directed movement toward the egg.

Speract, a decapeptide from Strongylocentrotus purpuratus sea urchin eggs, transiently stimulates a membrane guanylyl cyclase and activates a K(+)-selective channel that hyperpolarizes the sperm. Membrane potential recordings with fluorescent dyes in sperm flagellar vesicles were used to determine if calmodulin participates in the signal transduction induced by speract. The vesicle hyperpolarization induced by speract was inhibited by the calmodulin antagonists: trifluoperazine, mastoparan; N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide, (W-7); and N-(6-Aminohexyl)-1-naphthalenesulfonamide, (W-5). Since that inhibition occurred at concentrations at which calmodulin action is inhibited by these compounds, the overall findings suggested that calmodulin could be involved in the speract response. The speract response was Ca(2+)-independent however. Ten millimolar EGTA does not inhibit the hyperpolarization and 2 mM BAPTA only partially inhibited the response. High concentrations of calmodulin-dependent kinase II and phosphatase inhibitors did not alter the response of the flagella vesicles to speract. Taken as a whole, these results indicate that the speract-induced hyperpolarization involves the participation of calmodulin in a Ca2+ independent manner.

Identification of voltage-dependent Ca2+ channels in sea urchin sperm.

Functional evidence indicates that voltage-dependent Ca2+ (Cav) channels participate in sea urchin sperm motility and the acrosome reaction (AR), however, their molecular identity remains unknown. We have identified transcripts for two Ca2+ channel alpha1 subunits in sea urchin testis similar in sequence to Cav1.2 and Cav2.3. Antibodies against rat Cav1.2 and Cav2.3 channels differentially label proteins in the flagella and acrosome of mature sea urchin sperm. The Cav channel antagonists nifedipine and nimodipine, which inhibit the AR, diminish the intracellular Ca2+ elevation induced by a K+-induced depolarization in valinomycin-treated sperm. These findings reveal that Cav1.2 and Cav2.3 channels could participate in motility and/or the AR in sea urchin sperm.

A sustained increase in intracellular Ca(2+) is required for the acrosome reaction in sea urchin sperm.

The acrosome reaction (AR), necessary for fertilization in many species, requires an increase in intracellular Ca(2+) ([Ca(2+)](i)). In sea urchin sperm, the AR is triggered by an egg-jelly factor: the associated [Ca(2+)](i) elevation lasts minutes and involves two Ca(2+) permeable channels. Both the opening of the second channel and the onset of the AR occur approximately 5 s after treatment with egg factor, suggesting that these events are linked. In agreement, removal of Ca(2+) from sea water or addition of Ca(2+) channel blockers at the time when opening of the second channel is first detected inhibits AR and causes a "rapid" (t(1/2) = 3--15 s) decrease in [Ca(2+)](i) and partial inhibition of the intracellular pH change associated with the AR. Simultaneous addition of NH(4)Cl and either EGTA, Co(2+), or Ni(2+) 5 s after egg factor prevents the partial inhibition of the evoked pH(i) change observed but does not reverse AR inhibition. Therefore, the sustained increase in [Ca(2+)](i) caused by the second Ca(2+) channel is needed for the sperm AR. Experiments with agents that induce capacitative Ca(2+) uptake (thapsigargin and cyclopiazonic acid) suggest that the second channel opened during the AR could be a store-operated Ca(2+) channel.

Participation of a K(+) channel modulated directly by cGMP in the speract-induced signaling cascade of strongylocentrotus purpuratus sea urchin sperm.

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.