Intracellular calcium release at fertilization in the sea urchin egg

Abstract

Fertilization or ionophore activation of Lytechinus pictus eggs can be monitored after injection with the Ca-sensitive photoprotein aequorin to estimate calcium release during activation. We estimate the peak calcium transient to reach concentrations of 2.5–4.5 μM free calcium 45–60 sec after activation and to last 2–3 min, assuming equal Ca²⁺ release throughout the cytoplasm. Calcium is released from an intracellular store, since similar responses are obtained during fertilization at a wide range of external calcium concentrations or in zero-calcium seawater in ionophore activations. In another effort to estimate free calcium at fertilization, we isolated egg cortices, added back calcium quantitatively, and fixed for observation with a scanning electron microscope. In this way, we determined that the threshold for discharge of the cortical granules is between 9 and 18 μM Ca²⁺. Therefore, the threshold for the in vitro cortical reaction is about five times the amount of free calcium, assuming equal distribution in the egg. This result suggests that transient calcium release is confined to the inner subsurface of the egg.

Fig 1

Response to fertilization. Sperm addition at arrow. Time base is horizontal bar equal to 1 min. (A) Best aequorin emission: 25 eggs injected, 12 successfully fertilized. Vertical bar: 2 nA. (B) Typical aequorin emission: 27 eggs injected, 7 successfully fertilized. Vertical bar: 1 nA. In all figures, lower left horizontal bar is zero light level. All experiments conducted at 18°C.

Fig. 2

Response to ionophore activation. Addition of 40 μM A23187 at arrow. Vertical bar: 1 nA. Horizontal bar: 1 min. (A) Normal seawater containing 11 mM CaCl₂; out of 27 eggs injected, 6 successfully elevated fertilization membranes. (B) Zero-calcium seawater (0-Ca SW) with 2 mM EGTA added. Twenty-four eggs injected; fertilization membranes are not stable in zero-calcium seawater and could not be scored.

Fig. 3

Responses to fertilization and changes in Ca²⁺ concentration. Horizontal bar: 1 min; vertical bar: 1 nA. Unless otherwise noted, sperm addition at first arrow. (A) Artificial seawater (ASW) containing 460 mM NaCl, 55 mM MgCl₂, 11 mM CaCl₂, 10 mM KCl, and 5 mM NaHCO₃, pH 8, changed at second arrow to identical solution during successful fertilization of 7 eggs out of 27 injected. (B₁) ASW changed to 0-Ca SW with 2 mM EGTA at arrow while monitoring 24 injected unfertilized eggs. (B₂) ASW changed to 0-Ca SW containing 2 mM EGTA during the successful fertilization of 17 eggs out of 22 injected. (C₁) ASW changed to 55 mM Ca SW at arrow while monitoring 16 injected unfertilized eggs. (C₂) ASW changed to 55 mM Ca SW during fertilization of 31 injected eggs. Fertilization was not scored. Peak current at second arrow was 9 nA.

Fig. 4

Calibration of aequorin emission versus free Ca²⁺ on the basis of 10 injected eggs (see text). One nanoampere corresponds to 4 × 10⁻⁵ part injected aequorin exhausted per second. Anode voltage: 870 V. Temperature is 18°C.

Fig. 5

Aequorin emission from seven injected eggs poisoned with 1 mM NEM added at arrow. Blank spot in record is where tape reels were changed. Horizontal bar: 10 min. Vertical bar: 1 nA.

Fig. 6

Scanning electron micrograph of isolated inner surfaces before and after discharge with known concentrations of Ca²⁺. (a₁) Inner surface in homogenization buffer at less than 75 nM Ca²⁺. 700 ×. (a₂) Higher magnification of a, at 5000 × with individual 1.1-μm cortical granules intact, (b₁) Inner surface after washing and fixing in a Ca-EGTA buffer with 9 μM free Ca²⁺, which was subthreshold for cortical granule discharge. (b₂) Higher magnification of b,. 5000 ×. (c₁) Discharge of the cortical granules rapidly occurs in higher Ca²⁺ concentrations. Here, free Ca²⁺ = 18 μM. 700 ×. (c₂) Higher magnification of c₁. 5000 ×. (d₁) The image of surfaces exposed to even higher Ca²⁺ concentrations does not differ much from those at threshold concentrations. Free Ca²⁺ = 27.5 μM. 700 ×. (d₂) Higher magnification of d₁. 5000 ×.