Real-Time Measurements of the Interactions between Fluorescent Speract and Its Sperm Receptor

AbstractLytechinus pictus sea urchin sperm express receptors for speract, a sperm-activating peptide derived from the homologous egg jelly coat. We found that the fluorescence of fluorophore-labeled, active, speract analogs is quenched upon receptor binding. This property allowed us to perform real-time measurements of speract–receptor interactions using intact sperm and to determine, for the first time, their association (kon) and dissociation (koff) rate constants. The high kon (2.4 × 107 M−1 s−1) and low koff (4.4 × 10−6 s−1 (95%) and 3.7 × 10−4 s−1 (5%)) can account for the sperm response to picomolar concentrations of speract. We also examined the influence of extracellular ions on speract–receptor interactions using the fluorescence quenching method described in this study. The association rate of speract to the receptor is dramatically reduced in Na+-free seawater (NaFSW), divalent cation-free seawater (DCFSW), and high-K+ seawater (HKSW). In seawater speract induces an increase in intracellular pH (pHi), while it is unable to do so in either NaFSW or HKSW. To test if the lack of this pHi change causes the reduction in the speract association rate, pHi was increased with NH4Cl (10 mM) at the time labeled speract was added. Interestingly, this procedure completely (in HKSW) or partially (in NaFSW and DCFSW) restored the speract association rate to its receptor. These findings indicate that an increase in sperm pHi positively affects the receptor binding activity for this peptide and may partially explain the positive binding cooperativity displayed by the speract receptor

Modular analysis of the control of flagellar Ca2+-spike trains produced by CatSper and CaV channels in sea urchin sperm

Intracellular calcium ([Ca2+]i) is a basic and ubiquitous cellular signal controlling a wide variety of biological processes. A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of [Ca2+]i spikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelope and temporal organisation of the [Ca2+]i-spike trains. The signalling network comprises an initial membrane hyperpolarisation produced by an Upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules leading to intraflagellar pH (pHi), voltage and [Ca2+]i fluctuations. The Upstream module outputs were fitted to kinetic data on cGMP activity and early membrane potential changes measured in bulk cell populations. Two candidate modules featuring voltage-dependent Ca2+-channels link these outputs to the downstream dynamics and can independently explain the typical decaying envelope and the progressive spacing of the spikes. In the first module, [Ca2+]i-spike trains require the concerted action of a classical CaV-like channel and a potassium channel, BK (Slo1), whereas the second module relies on pHi-dependent CatSper dynamics articulated with voltage-dependent neutral sodium-proton exchanger (NHE). We analysed the dynamics of these two modules alone and in mixed scenarios. We show that the [Ca2+]i dynamics observed experimentally after sustained alkalinisation can be reproduced by a model featuring the CatSper and NHE module but not by those including the pH-independent CaV and BK module or proportionate mixed scenarios. We conclude in favour of the module containing CatSper and NHE and highlight experimentally testable predictions that would corroborate this conclusion

Tuning sperm chemotaxis by calcium burst timing

AbstractMarine invertebrate oocytes establish chemoattractant gradients that guide spermatozoa towards their source. In sea urchin spermatozoa, this relocation requires coordinated motility changes initiated by Ca2+-driven alterations in sperm flagellar curvature. We discovered that Lytechinus pictus spermatozoa undergo chemotaxis in response to speract, an egg-derived decapeptide previously noted to stimulate non-chemotactic motility alterations in Strongylocentrotus purpuratus spermatozoa. Sperm of both species responded to speract gradients with a sequence of turning episodes that correlate with transient flagellar Ca2+ increases, yet only L. pictus spermatozoa accumulated at the gradient source. Detailed analysis of sperm behavior revealed that L. pictus spermatozoa selectively undergo Ca2+ fluctuations while swimming along negative speract gradients while S. purpuratus sperm generate Ca2+ fluctuations in a spatially non-selective manner. This difference is attributed to the selective suppression of Ca2+ fluctuations of L. pictus spermatozoa as they swim towards the source of the chemoattractant gradient. This is the first study to compare and characterize the motility components that differ in chemotactic and non-chemotactic spermatozoa. Tuning of Ca2+ fluctuations and associated turning episodes to the chemoattractant gradient polarity is a central feature of sea urchin sperm chemotaxis and may be a feature of sperm chemotaxis in general

Real-time analysis of the role of Ca2+ in flagellar movement and motility in single sea urchin sperm

Eggs of many marine and mammalian species attract sperm by releasing chemoattractants that modify the bending properties of flagella to redirect sperm paths toward the egg. This process, called chemotaxis, is dependent on extracellular Ca2+. We used stroboscopic fluorescence imaging to measure intracellular Ca2+ concentration ([Ca2+]i) in the flagella of swimming sea urchin sperm. Uncaging of cyclic GMP induced Ca2+ entry via at least two distinct pathways, and we identified a nimodipine-sensitive pathway, compartmentalized in the flagella, as a key regulator of flagellar bending and directed motility changes. We found that, contrary to current models, the degree of flagellar bending does not vary in proportion to the overall [Ca2+]i. Instead we propose a new model whereby flagella bending is increased by Ca2+ flux through the nimodipine-sensitive pathway, and is unaffected by [Ca2+]i increases through alternative pathways