Crosstalk between protein kinases A and C regulates sea urchin sperm motility

Summary Fertilization, a crucial event for species preservation, in sea urchins, as in many other organisms, requires sperm motility regulation. In Strongylocentrotus purpuratus sea urchins, speract, a sperm chemoattractant component released to seawater from the outer egg layer, attracts sperm after binding to its receptor in the sperm flagellum. Previous experiments performed in demembranated sperm indicated that motility regulation in these cells involved protein phosphorylation mainly due to the cAMP-dependent protein kinase (PKA). However, little information is known about the involvement of protein kinase C (PKC) in this process. In this work, using intact S. purpuratus sea urchin sperm, we show that: (i) the levels of both phosphorylated PKA (PKA substrates) and PKC (PKC substrates) substrates change between immotile, motile and speract-stimulated sperm, and (ii) the non-competitive PKA (H89) and PKC (chelerythrine) inhibitors diminish the circular velocity of sperm and alter the phosphorylation levels of PKA substrates and PKC substrates, while the competitive inhibitors Rp-cAMP and bisindolylmaleimide (BIM) do not. Altogether, our results show that both PKA and PKC participate in sperm motility regulation through a crosstalk in the signalling pathway. These results contribute to a better understanding of the mechanisms that govern motility in sea urchin sperm.

Mechanical induction of oscillatory movement in demembranated, immotile flagella of sea urchin sperm at very low ATP concentrations

ABSTRACTOscillation is a characteristic feature of eukaryotic flagellar movement. The mechanism involves the control of dynein-driven microtubule sliding under self-regulatory mechanical feedback within the axoneme. To define the essential factors determining the induction of oscillation, we developed a novel experiment by applying mechanical deformation of demembranated, immotile sea urchin sperm flagella at very low ATP concentrations, below the threshold of ATP required for spontaneous beating. Upon application of mechanical deformation at above 1.5 µmol l−1 ATP, a pair of bends could be induced and was accompanied by bend growth and propagation, followed by switching the bending direction. For an oscillatory, cyclical bending response to occur, the velocity of bend propagation towards the flagellar tip must be kept above certain levels. Continuous formation of new bends at the flagellar base was coupled with synchronized decay of the preceding paired bends. Induction of cyclical bends was initiated in a constant direction relative to the axis of the flagellar 9+2 structure, and resulted in the so-called principal bend. In addition, stoppage of the bending response occasionally occurred during development of a new principal bend, and in this situation, formation of a new reverse bend did not occur. This observation indicates that the reverse bend is always active, opposing the principal bend. The results show that mechanical strain of bending is a central component regulating the bend oscillation, and switching of the bend direction appears to be controlled, in part, by the velocity of wave propagation.