KCl-Induced Calcium Rise in Squid Eggs: Measurement of Fura-2 Fluorescence.

Abstract

Sperm break the block to development in unfertilized eggs by somehow inducing a large increase in intracellular free Ca2’ ([Ca2’li). The mechanisms and sources for the increase in [Ca2’]i have been hypothesized to be different in deuterostome and protostome eggs (1). A release of intracellular Ca*+ upon fertilization plays a major role in the regenerative and explosive increase in [Ca2+li in deuterostome eggs. By contrast, extracellular Ca*+ entry may be more important for the prolonged increase in [Ca2+]i in protostome eggs. However, solid evidence substantiating this hypothesis in protostome eggs is limited. Squid eggs are unique in their size, mode of development, and phylogenetic position among protostomes (2). We have used these eggs to 1) investigate the existence of voltage-dependent calcium channels in unfertilized egg membranes, 2) establish the methodology of the fura2 fluorescence measurement, and 3) correlate KCl-induced Ca*+ rise with egg activation. Squid (Loligo pealei) were provided by the Marine Resources Department of the Marine Biological Laboratory during July and August. Clear, mature eggs were collected from the oviduct of female squid and washed several times with aerated millipore filtered artificial seawater (ASW). These eggs were put on coverslips, coated with poly-1-lysine, and maintained in ASW. A dual-excitation photometer (Deltascan; Photon Technology Inc.) was combined with the furatechnique (3) to measure [Ca’+]i. Eggs were loaded with 10 PLM fura-2/AM [the membrane-permeable form, dissolved in dimethyl sulfoxide (DMSO)] for 3 h at room temperature, in the dark. Subsequently, the eggs were thoroughly washed with ASW and then mounted on the stage of an epifluorescence inverted microscope (Nikon diaphot). The fluorescence ratio obtained with two excitation wavelengths, i.e., 350 nm and 380 nm, was used to monitor the relative [Ca’+]i. EGTA (1 mM) was added to nominally Ca’+-free ASW to make Ca”-free artificial seawater (CaFASW). The appearance of polar bodies 1 h after the addition of either KC1 or sperm was taken as indicating that the squid eggs had been activated or fertilized. All experiments were executed with batches of eggs exhibiting less than 6% spontaneous activation. Intracellular furaloading was reproducible in every batch of squid eggs. High concentration of extracellular K+ applied to squid eggs maintained in ASW increased [Ca2+li; two patterns of this effect were observed (Fig. 1). First, rapid increase in [Ca”]i induced by 15 m&f KC1 was seen in eight eggs (fast pattern). The increase in [Ca2+li was maximally developed within 1 min and was maintained at this enhanced level for more than 10 min after the addition of KCl. Moreover, this increased [Ca”]i did not tend to decay subsequently. In another group of eggs, 15 mil4 KC1 only induced a slow increase in [Ca2+]i (n = 5) (slow pattern). Here, [Ca’+]i gradually rose over 30 min after the application of KCl. Only the slow pattern of increase in [Ca2+li could be induced by 15 n-n44 KC1 in eggs that were exposed to CaFASW. Furthermore, the amplitude of KCI-induced slow increase in [Ca2+]i in eggs bathed in CaFASW was approximately 37% (n = 3) of that in eggs bathed in ASW. These results suggest that a high concentration of extracellular K+ can increase [Ca2+]i by acting on both extracellular and intracellular calcium pools with extracellular pool as the major source. In eggs exposed to ASW without a KC1 challenge, neither pattern of [Ca2+li change could be observed. After correcting for the spontaneous activation, we found that 26 + 6% and 22 + 6% of eggs, respectively loaded and not loaded with fura-2, were fertilized 60 min after the addition of squid sperm. DMSO alone did not affect the percentage of fertilization of eggs. Morphological observation also revealed that 15 mA4