Source during and Sinks for the Calcium Released Fertilization of Single Sea Urchin Eggs

The source and sinks for the intracellular calcium released during fertilization were examined in single eggs from the sea urchin, Arbacia punctulata. Single eggs were microinjected with the calcium photoprotein, aequorin. The calcium-aequorin luminescence was measured with a microscope-photomultiplier or observed with a microscope-image intensifier-video system. In the normal egg a propagated release has been observed. The source of the calcium was investigated in the organelle-stratified centrifuged egg and by the use of mitochondrial uncouplers. In the organelle-stratified centrifuged egg, the calciumaequorin luminescence was found to originate from the clear zone. The principal constituent of the clear zone is the endoplasmic reticulum. Other potential sources of calcium are the mitochondria. Their contribution to the calcium transient was investigated by exposure of aequorin-injected eggs to mitochondrial uncouplers either before or after fertilization. There was no calcium released from the mitochondria before fertilization. A very large calcium store was released from the mitochondria after fertilization. interestingly, eggs fertilized in the presence of uncouplers showed no increase in the calcium-aequorin luminescence over untreated eggs. Apparently, in the absence of mitochondrial uptake, other sinks for calcium with affinity and capacity similar to the mitochondria exist, but their nature is unknown. We suggest that the endoplasmic reticulum is the source of the intracellular calcium released upon fertilization and that the mitochondria are the principal sink. The results are discussed with regard to the metabolic activation of the egg. A transient increase in cytoplasmic free calcium occurs during fertilization in sea urchin and other eggs (12, 13, 15, 20, 28, 43, 48). The increase in free calcium is thought to be essential for structural and metabolic changes attendant upon fertilization (18, 21, 22). We previously have shown that the free calcium increases to -1 uM at ~23 s after the onset of the fertilization potential in the egg from the sea urchin, Arbacia punctulata. The increase in cytoplasmic free calcium originates at the point of insemination and rapidly expands across the egg within 6-9 s (13, 15). In these studies we have used the calcium-specific photoprotein, aequorin, as an indicator of cytoplasmic free calcium. The calcium-aequorin luminescence was measured with a microscope-photomultiplier or spatially resolved with a microscope-image intensifier-video system. 1522 We have investigated the sources of and sinks for the intracellular calcium released during fertilization of single eggs of the sea urchin, A. punctulata. The egg from this species of sea urchin is remarkable in that, when centrifuged, the organelles will stratify within the living, fertilizable egg according to their specific gravity (1, 22). From centripetal to centrifugal poles the strata as seen in the light microscope include the lipid cap, clear zone, granular zone, and pigment granules. The cortex remains undisplaced. Ultrastructural studies have better defined the constituents of each stratum (1). Although there is imperfect separation of the organelles, the centrifuged egg provides a way to examine the subcellular source of and sink for the released calcium under physiologic conditions in a single, living egg. If aequorin is used as the indicator of free calcium, then the luminescence will be observed originating THE JOURNAL OF CELL BIOLOGY . VOLUME 100 MAY 1985 1522-1527 © The Rockefeller University Press • 0021-9525/85/05/1522•06 $1.00 on Jauary 4, 2018 jcb.rress.org D ow nladed fom from the stratum containing the source. The source of released calcium and the sink that buffers the cytoplasmic free calcium concentration in the sea urchin egg and other stimulus-responsive cells are of widespread interest. Much recent work has focused on two cellular components that are thought to regulate cytoplasmic free calcium: endoplasmic reticulum and mitochondria (4, 5, 26, 38, 42). In the centrifuged egg, the endoplasmic reticulum is the principal constituent of the clear zone (l). The mitochondria are scattered throughout the egg with an increased density in the lipid cap and between the clear and granular zones (1, 12, 22). In this study we report direct observations of the calciumaequorin luminescence during fertilization of single, aequorin-injected, centrifugally stratified A. punctulata eggs. We find that the luminescence originates principally from the clear zone. This suggests that the endoplasmic reticulum is the source of the released calcium. A mitochondrial contribution to the released calcium has been excluded by the discovery that no calcium can be released with uncoupling agents prior to fertilization. On the other hand, a very large store of calcium can be released from the mitochondria after fertilization. This indicates that the mitochondria are quite effective in buffering the cytoplasmic free calcium. Additional studies suggest that other sinks exist which are capable of significant calcium buffering. MATERIALS AND METHODS Gametes were obtained from Arbacia punctulata by electrical stimulation or intracoelemic injection of 0.5 M KCI (9). Eggs were washed twice in artificial or filtered natural sea water. Eggs were immobilized, compressed and slightly flattened, between parallel coverslips in a chamber designed for microinjection (27). Freshly diluted sperm were added to the chamber in the dark through a micropipette positioned near the egg. Sperm have access to the egg in a restricted equatorial distribution. Solutions of mitochondrial uncouplers in sea water (41 uM carbonyl cyanide-p-trifluormethoxy phenylbydrazone [FCCP] ~ and 5 mM 2,4-dinitrophenol) were similarly added through micropipettes positioned near the egg. Experiments were conducted at room temperature (18-20"C). Eggs were organdie-stratified by placing 0.5 ml of a suspension of washed eggs in sea water atop a 0.5-ml cushion of 1 M sucrose in an Eppendorf centrifuge tube. This was then centrifuged in either an Eppendorf microfuge or an inexpensive kitchen blender modified to operate as a centrifuge under the same conditions. Centrifuge times varied with the batch of eggs from 1-3 rain. The stratified eggs were removed from the sea water-sucrose cushion interface and loaded into the injection chamber which was then filled with sea water. With care it is possible to microinject the stratified eggs without disrupting the stratification (12). Aequorin or acetylated aequorin, a sensitive and specific calcium photoprotein (6, 39, 40), was pressure-injected into a single 75-~tm-diam A. punctulata egg (either normal or stratified) for each experiment; about 14 pl of the aequorin solution was injected (10 mg/ml in 50 mM HEPES, 0.2 mM EGTA, pH 6.97.0). It was found that HEPES buffer was tolerated better on injection into the cells than others that were tried (12). The inclusion of EGTA in the injection buffer protected the aequorin from accidental contamination and led to light signals higher than those from chelator-free preparations (12). The acetylated aequorin was used in later studies. It produces a greater luminescence than the native protein (40). The final concentration of aequorin in the injected A. punctulata egg was ~13 ~M. The aequorin distribution in the microinjecled eggs was assumed to approximate the distribution offluorescein isotbiocyanatelabeled albumin which was shown to distribute in the aqueous cytoplasm (12). For the detection of the calcium-aequorin luminescence, light collected by an "objective" (Nikon 24×, 1.15 NA condenser;, Zeiss 25x, 0,8 NA; Zeiss 25x, 0.6 NA, Leitz 50x, 1,0 NA) was directed to either a photomultiplier or an image intensifier. An EMI 6256 photomultiplier was operated at -1100 V and had a dark current o f 1 0 pA. The output was amplified with a Kiethley electrometer, and the current was recorded on a Gould chart recorder (Gould Inc., Rolling Meadows, 1L). The output of the EMI four-stage, magnetically Abbreviation used in this paper: FCCP, carbonyl cyanide-p-tr/fluormethoxy phenylhydrazone. focused image intensifier (9912) was photographically integrated for various periods on Polaroid 3000 ASA film during the initial studies with the native aequorin. In later studies with both the native and acetylated aequorin, the image intensifier output was observed in real time with a Dage silicon-intensified target vidicon (Dage-MTI Inc., Wabash, MI) and recorded on videotape. The video record was photographically integrated for various periods from the video monitor on Plus-X film. The microscopic system also included a Zeiss epifluorescence attachment for examination of the pyridine nucleotide fluorescence (8, 16, 17). Excitation is at 365 nm with fluorescence at 450 nm. Fig. 1 presents a schematic of the experimental system which is described in detail elsewhere (35-37). Photographs of the eggs before and after fertilization were taken by conventional photomicrography. Analysis of both the temporal and spatial dynamics of the calcium transient was accomplished by photographing the video monitor during video playback for successive I-s intervals with ~Y4 s between exposures for automatic advancement of the film The photographs could be subsequently analyzed by densitometry.