Figure 1.

ZP3 produces Ca²⁺_i transients that are detected with mag-fura2. (A) Dye (λ_EX, 360 nm; λ_EM, 510 nm) distributes throughout sperm and permits ratiometric image acquisition from all regions. (B) Ca²⁺_i responses to ZP3 treatment in the absence (○) and the presence (●) of PN200–110. Cells were treated with buffer (b) or with 1 μM PN200–110 (pn) for 10 min before addition of ZP3 (10 μg/ml; bar). The rising phase of the ZP3-dependent Ca²⁺_i response in the absence of channel antagonists is approximated by the expression Ca²⁺_i = e^t/τ, where t and τ represent the time and the time constant, respectively (—). In this cell, τ = 12.7 msec. The initial portion of the relaxation trace is approximated by the expression Ca²⁺_i = ae^−t/τ, where a designates the maximal response (- - -) and where a = 9.11 μM and τ = 40.9 msec. (C) Peak Ca²⁺_i responses to ZP (40 μg/ml) or to ZP3 (10 μg/ml) are inhibited by La³⁺ (○) and by Cd²⁺ (●). Data points represent the means (±SD) of observations on 4–7 different cells and are approximated by the expression R = (100 ∗ IC₅₀)/(IC₅₀ + D). R is observed response; D is antagonist concentration; and IC₅₀ is the drug concentration producing 50% inhibition. Derived IC₅₀ values were La³⁺, 171 ± 63 μM; and Cd²⁺, 652 ± 134 μM. Results using ZP3 and ZP are indistinguishable and are pooled for presentation. (D) ZP3-dependent Ca²⁺_i transients are inhibited by LVA current antagonists. Pimozide was added at 15 min of a 120-min incubation in a capacitating medium whereas PN200–110 and Ni²⁺ were added at 90 min. After incubation for 120 min, sperm were treated with ZP3. Data represent peak Ca²⁺_i values (mean ± SD). The fraction of cells in each treatment group that exhibit transients is indicated. Total cells were: buffer, 12; fetuin (40 μg/ml), 14; ZP (40 μg/ml), 26; ZP2 (40 μg/ml), 14; ZP3 (10 μg/ml), 37; +PN200–110 (0.1 μM), 9; +PN200–110 (1 μM), 11; +pimozide (0.1 μM), 12; +pimozide (1 μM), 16; +Ni²⁺ (5 μM), 8; +Ni²⁺ (50 μM), 7.