Ca(2+) entry through store-operated channels in mouse sperm is initiated by egg ZP3 and drives the acrosome reaction

Abstract

Fertilization occurs after the completion of the sperm acrosome reaction, a secretory event that is triggered during gamete adhesion. ZP3, an egg zona pellucida glycoprotein, produces a sustained increase of the internal Ca(2+) concentration in mouse sperm, leading to acrosome reactions. Here we show that the sustained Ca(2+) concentration increase is due to the persistent activation of a Ca(2+) influx mechanism during the late stages of ZP3 signal transduction. These cells also possess a Ca(2+) store depletion-activated Ca(2+) entry pathway that is open after treatment with thapsigargin. Thapsigargin and ZP3 activate the same Ca(2+) permeation mechanism, as demonstrated by fluorescence quenching experiments and by channel antagonists. These studies show that ZP3 generates a sustained Ca(2+) influx through a store depletion-operated pathway and that this drives the exocytotic acrosome reaction.

Figure 1

ZP3 produces a sustained Ca²⁺_i increase in capacitated mouse sperm. (A) Resting Ca²⁺_i values were determined before the addition of ZP3 (10 μg/ml). Similar results were obtained with ZP extracts containing ZP3 (40 μg/ml ZP; our unpublished results). Maximal Ca²⁺_i response rates (ΔCa²⁺_i/Δt) were determined from linear fits of the slopes of time courses (straight line) and corrected for agonist-independent rates. Rate values and the initial and final calculated Ca²⁺_i values are displayed here and in subsequent figures. (B) Control glycoproteins (10 μg/ml fetuin and mouse ZP2) failed to initiate robust Ca²⁺_i responses. (C) Robust Ca²⁺_i responses were initiated by ZP3 from oocytes but not by ZP from two-cell embryos (ZP_2C; 10 μg/ml). Data are representative of 122 cells treated with ZP3 and 9–34 cells treated with control glycoproteins.

Figure 2

Sustained Ca²⁺_i increases after ZP3 treatment require Ca²⁺_o. Sperm were loaded with fura 2 and incubated in a 1.7 mM Ca²⁺ medium. BAPTA and Ca²⁺ (5 mM) additions served to alternate Ca²⁺_o between >30 nM and 1.7 mM, respectively. ZP3 (10 μg/ml) was added as indicated. Traces are representative of 13–47 cells in these treatment groups. Similar results were obtained with the use of ZP extracts.

Figure 3

Mn²⁺ influx is activated by ZP3 but not by ZP2. Sperm were loaded with fura 2 and incubated in a Ca²⁺-depleted medium. Mn²⁺ influx was monitored by the quenching of fura 2 fluorescence (excitation wavelength, 360 nm). ZP3 or ZP2 (10 μg/ml) was added as indicated (bars). Times of 10 μM Mn²⁺ addition are indicated (arrows in traces A2–A5 and B2 and B3). Relative fluorescence emission data (F_n/F_i) are presented, where F_n and F_i are emission at n min and at initial time, respectively. (A) ZP3 produces a sustained Mn²⁺ influx. Similar results were obtained with the use of ZP extracts containing ZP3 (40 μg/ml). (B) ZP2 does not activate a Mn²⁺ influx pathway. Traces are from 8 different cells and are typical of responses in 11 cells treated with buffer (traces A1 and B1), 37 cells treated with ZP3 or ZP solutions (traces A2–A5), and 21 cells treated with ZP2 (traces B2 and B3).

Figure 4

Ni²⁺ inhibits Ca²⁺_i responses to ZP3. Ni²⁺ was added either 2 min before (○) or 15 s after ZP3 (). Response rates (ΔCa²⁺_i/Δt) were determined as described (Figure 1). (A) Data are means ± SD of observations on 7–22 cells and are fit to the expression I = (100 × IC₅₀)/(IC₅₀ + C), where I is the inhibition of response rates in the presence Ni²⁺ and C and IC₅₀ are the specific Ni²⁺ concentration and the concentration that inhibits Ca²⁺_i response by 50%, respectively. Calculated values of IC₅₀ were 26 μM for Ni²⁺ before ZP3 (black line) and 481 μM for Ni²⁺ after ZP3 (gray line). Letters (a and b) designate points that are illustrated by traces in B. (B) Montage of traces showing sperm treatment with 10 μg/ml ZP3 (●), with 1 mM Ni²⁺ added 15 s after ZP3 (), or with Ni²⁺ added 2 min before ZP3 (○).

Figure 5

Thapsigargin activates Ca²⁺ influx. Fura 2–loaded sperm were treated with 10 μM thapsigargin (tp) in a <30 nM Ca²⁺ medium, and 1.7 mM Ca²⁺_o was added. (A–E) Sustained increases of Ca²⁺_i were observed in the presence of Ca²⁺_o and were curtailed by 5 mM BAPTA_o or 100 μM La³⁺. (F) Sperm were incubated in <30 nM Ca²⁺_o containing 10 μM Mn²⁺_o. Thapsigargin activated Mn²⁺ influx in fura 2/BAPTA_i-loaded sperm, resulting in quenching of fura 2 fluorescence. Fluorescence quenching data are expressed as the value F_n/F_i, as described in Figure 3. Representative traces were chosen from the following numbers of cells: (A) 71, (B) 49, (C) 44, (D) 19, (E) 17, and (F) 9.

Figure 6

Thapsigargin and ZP3 activate a similar cation permeation pathway in sperm. Fura 2–loaded sperm were incubated in a <30 nM Ca²⁺_o medium, and Ca²⁺-independent dye fluorescence was monitored as described in Figure 3. Mn²⁺ (1 μM), thapsigargin (tp; 10 μM) and ZP3 (10 μg/ml) were added (dashed lines), and fluorescence quenching rates were determined by the slopes of these traces. (A) Quenching rates ([F_x/F_i]/[t_x/t_i], where F_x and F_i are the fluorescence emission at x min [t_x] and at the initial time [t_i], respectively) were determined for individual sperm during a 2-min incubation in 1 μM Mn²⁺ and designated R_L (leak rate). Quenching rates during experimental treatment (R_X) were determined during a 10-min incubation with thapsigargin or with ZP3 or by the addition of one reagent followed 3 min later by the second reagent. Results are expressed as the relative quench rate (R_X/R_L) and are the mean of observations on 12–39 sperm. (B) A fluorescence trace showing the effects of thapsigargin and ZP3 on Mn²⁺-dependent quenching. Data from this and other cells were used to construct panel A. (C) Ni²⁺_o inhibits thapsigargin-induced Ca²⁺_i responses. Thapsigargin triggered Ca²⁺_i increases upon addition of Ca²⁺_o. Addition of Ni²⁺ 15 s after Ca²⁺_o inhibited this response in a concentration-dependent manner (▿). Data were fit to the expression described in Figure 4, and the derived IC₅₀ value was 517 μM. For comparison, fit curves for the inhibition of the early (black dashed line) and late (gray dashed line) phases of ZP3-evoked Ca²⁺_i responses are redrawn from Figure 4A. The letter (a) designates the point that is used in the traces to the right. These traces are from two sperm in which thapsigargin triggered Ca²⁺_i responses in the absence (▾) and in the presence (▿) of 1 μM. Ni²⁺_o.

Figure 7

AN1043 inhibits the thapsigargin-dependent Ca²⁺_i regulator of sperm but not the LVA channel. (A) The Ca²⁺ permeation pathway of sperm was activated by treatment with 10 μM thapsigargin (tp), followed by the addition of 1.7 mM Ca²⁺_o. AN1043 (10 μM) or DMSO was added 15 s after Ca²⁺_o. Initial and final Ca²⁺_i values are displayed, as are the maximum response rates. Traces are representative of observations on 31 cells treated with control medium and 23 cells treated with AN1043. (B) Dose-response relationship for the inhibition of sustained Ca²⁺_i by AN1043. Data points are means (±SD) of observations on 16–23 cells in AN1043 treatment groups and were normalized to a population of 31 cells that were treated with thapsigargin in the absence of AN1043. Data are fit to the same expression as in Figure 4, where the derived IC₅₀ value was 0.7 ± 0.2 μM. (C) Effects of AN1043 on the LVA current of mouse pachytene spermatocyte. Peak whole cell Ca²⁺ currents were recorded after test depolarizations to −30 mV from a holding potential of −90 mV. AN1043 (10 μM) was added as indicated. AN1043 reduced the peak current by 20% from an initial value of 245 pA. Data are representative of current recordings on six cells.

Figure 8

AN1043 inhibits ZP3 signal transduction. (A) Sperm were treated with 10 μM AN1043 (●), 10 μM nifedipine (▵), or DMSO vehicle (○) added either 5 min before (left) or 15 s after (right) the addition of 10 μg/ml ZP3 or 40 μg/ml ZP extracts. Acrosome reactions were determined during a 60-min incubation, and data were normalized according to the expression AR = (AR_i − AR₀)/(AR₀), where AR₀ and AR_i are the acrosome reaction levels before ZP/ZP3 addition and at i min. AR₀ was 21 ± 8 and 17 ± 5% when drugs were added before or after agonists, respectively. (B) Dose-response relationship for the inhibition of acrosome reactions after the addition of AN1043 15 s after ZP/ZP3. Acrosome reactions were determined 30 min after the addition of ZP/ZP3. Results are normalized according to the expression AR = 100 × [(AR_AN30 − AR₀)/(AR_30B − AR₀)], where AR₀, AR_AN30, and AR_30B are the levels of acrosome reactions at 0 min, at 30 min in the presence of AN1043, and at 30 min in the absence of AN1043, respectively. The derived IC₅₀ for inhibition of the acrosome reaction was 1.1 ± 0.2 μM. Data in A and B represent the means (±SD) of triplicate experiments, with 200 sperm examined at each time or concentration point in each experiment. (C) AN1043 inhibits the sustained Ca²⁺_i response that is activated by ZP/ZP3. Ca²⁺_i was monitored after the addition of either 10 μg/ml ZP3 or 40 μg/ml ZP. AN1043 or DMSO was added 15 s after the agonist. Response rates (ΔCa²⁺_i/Δt) were determined as described in Figure 1. (Left) Pooled response (means ± SD) of observations on 12–47 cells. Data were normalized according to the same expression as in B, in which data reflect rates (ΔCa²⁺/Δt) rather than acrosome reaction values. The derived IC₅₀ value for inhibition of the Ca²⁺_i response was 0.8 ± 0.1 μM. Letters above the bars refer to individual traces shown on the right. (Right) Representative traces for sperm treated with 0 μM (a), 1 μM (b), or 10 μM (c) AN1043.

Figure 9

Model of the ionic events of ZP3 signal transduction. (Top) ZP3 increases sperm Ca²⁺_i. (Bottom) This response may be due to the sequential activation of three Ca²⁺ channels. (Early events) ZP3 activation of a receptor (R) results in: 1) membrane depolarization (ΔV_M) and the subsequent Ca²⁺ influx (Ca_LVA) through LVA Ca²⁺ channels; and 2) the activation of phospholipase C (PLC), leading to the generation of IP3. (Intermediate events) IP3 activates IP3 receptors (IP3R) in the acrosomal membrane, leading to an efflux of Ca²⁺ from this internal store (Ca_S) into the cytosol. (Late events) Depletion of intracellular Ca²⁺ stores produces a signal (gating signal) that opens a store-operated Ca²⁺ channel (SOC) in the plasma membrane. SOC activation also requires Ca_LVA and may be further regulated by other ZP3-dependent events, such as increased internal pH. The resultant Ca²⁺ influx (Ca_SOC) drives acrosome reactions.