Aequorin-injected eggs of the medaka (a fresh water fish) show an explosive rise in free calcium during fertilization, which is followed by a slow return to the resting level.

No abstract

No abstract

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....

An interface of our microspectrofluorometer with an image processing system performs microspectrofluorometric measurements in living cells by digital image processing. Fluorescence spectroscopic parameters can be measured by digital image processing directly from microscopic images of cells, and are automatically normalized for pathlength and accessible volume. Thus, an accurate cytoplasmic "map" of various spectroscopic parameters can be produced . The resting cytoplasmic pH of fibroblasts (3T3 cells) has been determined by measuring the ratio of fluorescein fluorescence exited by two successive wavelengths (489 and 452 nm) . Fluorescein-labeled dextran microinjected into the cells is used as a pH indicator, since it is trapped in the cytoplasm but is excluded from the nucleus and other organelles . The average cytoplasmic pH is 6.83 (± 0.38). However, cytoplasmic pH exhibits a nonunimodal distribution, the lower mean pH being 6.74 (± 0.23). When 3T3 cells pinocytose medium containing fluorescein dextran, pinosomes peripheral to the nucleus exhibit a lower pH than those closer to the ruffling edge of the cell . The present image processing system is analyzed for linearity of detection, light scattering artifacts, signal to noise ratio, standard curves, and spatial resolution. The results obtained from digital image analysis are shown to be comparable to the results from standard microspectrofluorometry . We also discuss several other applications of this ratio imaging technique in cell biology.