Temporal Sequence and Spatial Distribution of Early of Fertilization in Single Sea Urchin Eggs Events

Measurements and observations of five early events of fertilization, singly and in pairs, from single sea urchin eggs have revealed the precise temporal sequence and spatial distribution of these events. In the Arbacia punctulata egg, a wave of surface contraction occurs coincident with membrane depolarization (t = 0). These two earliest events are followed by the onset of a rapid, propagated increase in cytoplasmic-free calcium at ~23 s as measured by calcium-aequorin luminescence. The luminescence reaches its peak value by 40 s after the membrane depolarization. The luminescence remains uniformly elevated for some time before its decay over several minutes. The onset of an increase in the pyridine nucleotide (NAD(P)H) fluorescence follows the membrane depolarization at ~51 s. The fertilization membrane begins its elevation in a wave-like fashion coincidentally with the increase in NAD(P)H fluorescence. Similar results are observed in the Lytechinus variegatus egg. The results suggest that while the increase in cytoplasmic-free calcium may be important for many changes occurring in the egg, the elevated-free calcium is not directly responsible for the propagated wave of cortical granule exocytosis. Fertilization initiates various physiological, biochemical, and structural changes within the egg that occur in a well-orchestrated manner both temporally and spatially. These events are requisite for normal embryonic development (8, 9). Included among the early events of fertilization are sperm-egg fusion, membrane depolarization, a surface contraction, an increase in cytoplasmic free calcium, the exocytosis of cortical granules, and a change in the pyridine nucleotide redox state. To some extent the sequence of these early events is known (8, 12, 19, 26). That we might better understand the significance and casual relationships among the various events, we have examined simultaneously several of the events that occur within single sea urchin eggs at fertilization. The use of single eggs allows for the precise determination of the sequence of events which is unobtainable from studies that employ a population of eggs. Specifically, we have examined the temporal and spatial relationships among the fertilization potential, surface contraction, increase in cytoplasmic free calcium, NAD(P)H fluorescence, and elevation of the fertilization membrane. The relationships among the various early events have been largely inferential although some work has been reported from single cells (12, 25, 26). We are principally concerned with when the increase in cytoplasmic-free calcium occurs relative to the other events. The increase in cytoplasmic-free calcium has been associated with sperm-egg fusion, initiation of the fertilization potential, the surface contraction, and, of course, the cortical reaction and fertilization membrane elevation (4, 8-11, 18, 20, 28, 29, 32). A precise determination of the temporal sequence of the early events in single eggs would help clarify the role, if any, for calcium in each of the other events. We find that the fertilization potential (t = 0) is followed by an increase in intracellular-free calcium beginning at 23 _+ 3 s (n = 3) and the redox state of the pyridine nucleotides becomes more reduced (fluorescent) at 51 + 7 s (n = 3) in the Arbacia punctulata egg. Further, we spatially resolve the distribution of both the calcium increase and the redox change and demonstrate that each occurs with a cytoplasmic distribution. Finally we note that the fertilization membrane begins THE JOURNAL OF CELL BIOLOGY VOLUME 99 NOVEMBER 1984 1647-1654 © The Rockefeller University Press • 0021-9525/84/11/1647/08 $1.00 1 6 4 7 on July 1, 2017 jcb.rress.org D ow nladed fom its elevation coincidentally with the change in the NAD(P)H redox state. Less extensive measurements from eggs of Lytechinus variegatus are qualitatively similar. MATERIALS AND METHODS Gametes were shed from .4. punctulata and L. variegatus by eleetric~ stimulation or intracoelemic injection of 0,5 M KCI. 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 (16). Freshly diluted sperm were added to the chamber in the dark through a micropipette positioned some distance away from the egg. Sperm have access to the egg in a restricted cquitorial distribution. Experiments were conducted at room temperature (18-20"C). Aequorio or acetylated aequorin, a sensitive and specific calcium-photoprotein (3, 29, 30), was pressure injected into a single 75-~m diam A. punctulata or 120-~m diam L. variegatus egg for each experiment. In A. punctulata 14 pl of the aequorin solution was injected (10 mg/ml in 50 mM HEPES, 0.2 mM EGTA, pH 6.9-7.0). It was found that HEPES buffer was tolerated better on injection into the cells than others that were tried. 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 (5). The acetylated aequorin was used in later studies where both the temporal and the spatial dynamics of the calcium transient were investigated as it has a higher photon yield than the native protein (30). The final concentration of aequorin in the injected A. punctulata egg was ~ 13 ~m. For the detection of the calcium-aequorin luminescense, light collected by an "objective ~ (Nikon ×24/1.15 NA condenser, Zeiss x25/0.8 NA, Zeiss x25/0.6 NA, Leitz x 50/1.0 NA) was directed to either a photomultiplier or an image intensifier. An EM16256 SA photomultiplier was operated at 1,100 V and had a dark current of ~ l0 pA. The output was amplifed with a Kiethley electrometer and the current recorded on a Gould chart recorder. The output of the EMI four-stage, magnetically focused image intensifier (9912) was observed in real time with a Doge silicon-intensified target (SIT) vidicon and recorded on video tape. Alternately the image tube output cold be directly photographed using high speed (ASA 10,000) Polaroid film. Electrophysiologic measurements were made with a 3 M KO-filled glass electrode (~100 Mohms) connected to a WPl M701 amplifier. The sea water bath in the Lucite chamber was grounded through a seawater-agar/Ag(C1)2/Ag wire. The 450 nm NAD(P)H fluorescence (5, 8) was stimulated by monochromatic 365 nm light attenuated with neutral density filters such that bleaching was reduced to <15% under continuous excitation (5). The fluorescence was measured with the photomultiplier tube noted above. 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 1-s intervals with ~1/4 s between exposures for automatic advancement of the film. The photographs could be subsequently analyzed by densitometry. Alternatively light guides have been placed against the video monitor overlying various positions of the image of the egg and the light directed to a photomultiplier. Fig. l presents a schematic of the experimental system and Fig. 2 demonstrates how the video images can be analyzed. FIGURE 1 System schematic. The experimental system consists of a microscope with a De-regulated Hg arc epi-fluorescence (EPI) attachment, low-noise/high-gain photomultiplier tube (PMT), and image intensifier tube (liT), silicon intensified target vidicon (SIT), and with various amplifiers (AMP) and recording devices to obtain temporal and spatial records of the fluorescent or luminescent changes occurring within a single egg. 1648 THE JOURNAL OF CELL BIOLOGY , VOLUME 99, 1984 Experimental Design: We wished to examine the temporal and spatial relationships of various early events of fertilization not all of which could be measured or observed simultaneously. The individual events have been studied in several hundred, single eggs. For studies of multiple events the membrane depolarization served as a temporal reference point. It is one of the earliest measureable changes that occurs at fertilization and it occurs over a far shorter period of time (~100 ms) relative to many other changes (13, 26). We have taken the membrane depolarization as t = 0. With care it is possible to first inject a single egg with aequorin and then follow with impalement by a microelectrode such that an undamaged membrane potential in the range of -75 to ~-125 mV is obtained (13, 14, 21). We were able to obtain simultaneous measurements of both the potential change and the simultaneous measurements of both potential change and the calcium transient that follows from three A. punctulata eggs. The results are summarized in Table I. Likewise, in a separate set of experiments we were able to obtain simultaneous measurements of both the membrane potential and the change in NAD(P)H fluorescence from three A. punctulata eggs. The results are summarized in Table II.