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

• The pHi increase induced by speract and cGMP precedes Ca2+ influx in the sperm flagellum.
• These pHi increases are independent of external Ca2+ and blocked by high KCl.
• The NH4Cl-induced alkalinization stimulates Ca2+ influx in the sperm head and flagellum.
• The NH4Cl-induced alkalinization increases Ca2+ first in the sperm flagellum.
• The pHi elevation in the flagellum regulates [Ca2+]i and possibly modulates sea urchin sperm swimming.

Speract, a peptide from the egg jelly coat of certain sea urchin species, modulates sperm motility through a signaling pathway involving several ionic fluxes leading to pHi and [Ca2+]i increases. [Ca2+]i oscillations in the flagellum regulate its beating pattern modulating sperm swimming. Recent evidence showed the importance of pHi in controlling Ca2+ influx and chemotaxis. However, spatio-temporal characterization of the flagellar pHi increase triggered by speract, and its correlation to that of [Ca2+]i is lacking. Here, we show for the first time in single sea urchin spermatozoa that the speract-induced flagellar pHi increase precedes and is independent of [Ca2+]i increase. Our results support a leading role of pHi in modulating the Ca2+ signals that govern sperm swimming.