Revisiting the Role of H+ in Chemotactic Signaling of Sperm

Abstract

Chemotaxis of sperm is an important step toward fertilization. During chemotaxis, sperm change their swimming behavior in a gradient of the chemoattractant that is released by the eggs, and finally sperm accumulate near the eggs. A well established model to study chemotaxis is the sea urchin Arbacia punctulata. Resact, the chemoattractant of Arbacia, is a peptide that binds to a receptor guanylyl cyclase. The signaling pathway underlying chemotaxis is still poorly understood. Stimulation of sperm with resact induces a variety of cellular events, including a rise in intracellular pH (pHi) and an influx of Ca2+; the Ca2+ entry is essential for the chemotactic behavior. Previous studies proposed that the influx of Ca2+ is initiated by the rise in pHi. According to this proposal, a cGMP-induced hyperpolarization activates a voltage-dependent Na+/H+ exchanger that expels H+ from the cell. Because some aspects of the proposed signaling pathway are inconsistent with recent results (Kaupp, U.B., J. Solzin, J.E. Brown, A. Helbig, V. Hagen, M. Beyermann, E. Hildebrand, and I. Weyand. 2003. Nat. Cell Biol. 5:109–117), we reexamined the role of protons in chemotaxis of sperm using kinetic measurements of the changes in pHi and intracellular Ca2+ concentration. We show that for physiological concentrations of resact (<25 pM), the influx of Ca2+ precedes the rise in pHi. Moreover, buffering of pHi completely abolishes the resact-induced pHi signal, but leaves the Ca2+ signal and the chemotactic motor response unaffected. We conclude that an elevation of pHi is required neither to open Ca2+-permeable channels nor to control the chemotactic behavior. Intracellular release of cGMP from a caged compound does not cause an increase in pHi, indicating that the rise in pHi is induced by cellular events unrelated to cGMP itself, but probably triggered by the consumption and subsequent replenishment of GTP. These results show that the resact-induced rise in pHi is not an obligatory step in sperm chemotactic signaling. A rise in pHi is also not required for peptide-induced Ca2+ entry into sperm of the sea urchin Strongylocentrotus purpuratus. Speract, a peptide of S. purpuratus may act as a chemoattractant as well or may serve functions other than chemotaxis.

Single cell imaging reveals that the motility regulator speract induces a flagellar alkalinization that precedes and is independent of Ca2+ influx in sea urchin spermatozoa

“…In this regard, our group found that speract from pM to nM concentrations increases pHi before [Ca 2+ ]i in S. purpuratus sperm populations [12,13]. Although earlier sperm population studies in Arbacia punctulata concluded that pM resact (the corresponding chemoattractant peptide) first elevates [Ca 2+ ]i and then pHi, and that this alkalinization did not influence chemotaxis [14], recently the authors of this study reported that alkalinization precedes the [Ca 2+ ]i elevation, and importantly, that it presumably stimulates…”

“…This intracellular alkalinization was proposed to mediate peptide receptor and guanylate cyclase inactivation through dephosphorylation [21,[23][24][25], though recent results question this proposal [26]. Sea urchin sperm population experiments had yielded contradictory results regarding their pHi responses to egg peptides [12][13][14]. Recent agreement regarding the kinetic precedence of pHi over [Ca 2+ ]i lead Seifert et al [6] to propose that the resact-induced pHi increase activates CatSper, the flagellar sperm specific pHi-activated Ca 2+ channel, regulating A. punctulata sperm chemotaxis.…”

Sperm-activating peptides in the regulation of ion fluxes, signal transduction and motility

“…The Kaupp group has reported that in A. punctulata and S. purpuratus the pHi increase at low SAP concentrations occurs after, not before, the [Ca 2+ ]i elevation and that the intracellular alkalinization is not due to activation of Na + /H + exchange induced by the hyperpolarization but probably caused by cytosolic proton consumption during GTP and ATP replenishment (Kaupp et al, 2006;Solzin et al, 2004;Strunker et al, 2006). These results are in conflict with those obtained by Nishigaki et al (2001Nishigaki et al ( , 2004.…”

A comparison of early molecular fertilization mechanisms in animals and flowering plants

“…Like resact, speract activates a cGMP-signaling pathway (Cook and Babcock 1993;Matsumoto et al 2003;Solzin et al 2004). However, the speract receptor, a 77 kDa plasma membrane receptor localized exclusively in the sperm flagellum, has been reported to be unrelated to guanylyl cyclase (Dangott et al 1989;Cardullo et al 1994).…”

“…However, the speract receptor, a 77 kDa plasma membrane receptor localized exclusively in the sperm flagellum, has been reported to be unrelated to guanylyl cyclase (Dangott et al 1989;Cardullo et al 1994). The speract-induced motility changes in S. purpuratus sperm closely resemble those of other marine species whose sperm undergo chemotaxis, but attempts have failed to demonstrate its chemotactic activity (Solzin et al 2004;Wood et al 2007). It was suggested that resact, speract and other SAPs function through a common signal transduction pathway to stimulate and orient sperm motility, and that this signal involves changes in intracellular pH, in the concentrations of cGMP, cAMP as well as Na + and Ca 2+ ions, in membrane potential and in the phosphorylation pattern of several proteins (Ward and Kopf 1993;Cook et al 1994;Darszon et al 2005Darszon et al , 2006Wood et al 2007).…”