Fig. 2. Change of [Ca²⁺]_i in hypotonic or hypertonic condition indicated by fluo-3 AM. Sperm were incubated with 500 µmoll^-1 fluo-3 AM in ASP (artificial seminal plasma) for 2 h. (A,B) Approximately 90% of sperm showed movement after dilution in hypotonic conditions: NaCl 50 mmoll^-1 + CaCl₂ 5 mmoll^-1 (A) or NaCl 50 mmoll^-1 + EGTA 5 mmoll^-1 (B). Confocal micrographs were taken approximately 30 min after the onset of activation, when almost all sperm had stopped moving. (C,D) In hypertonic conditions (300 mmoll^-1 NaCl), sperm did not move even in the presence of Ca²⁺ (D) and [Ca²⁺]_i was not increased, as indicated by the absence of fluorescence. Upper panels, phase contrast micrographs; lower photos, fluorescence micrographs. (E) Diagram of sleeve structure expanded in hypotonic conditions (as in A and B) but was shrunk in hypertonic conditions (as in C and D). Bars, 20 µm.