Temporal sequence and spatial distribution of early events of fertilization in single sea urchin eggs.

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....

Redox Control of Exocytosis

“…GRX1 has previously been proposed to play a role in exocytosis on basis of its immunohistochemical localization in synaptic terminals in the neurohypophysis (26), and an increased NADPH-to-NADP ϩ ratio has been linked to the intense wave of exocytosis of cortical granules oocyte fertilization (36). Redox-regulated posttranslational modification of exocytosis-regulating t-SNARE proteins has been proposed to result in an increased rate of maturation, or priming, of secretory vesicles in yeast (37).…”

Transitioning from egg to embryo: Triggers and mechanisms of egg activation

“…Fertilization ultimately causes a rise in intracellular Ca 2ϩ that starts from the point of sperm entry and spreads in a wave-like manner across the entire egg (Gilkey et al, 1978;Eisen et al, 1984;Sardet et al, 2006). This elevated Ca 2ϩ originates either from the external environment, from internal cellular stores, or from some combination of the two.…”

Accumulation of fluorescently labeled actin in the cortical layer in sea urchin eggs after fertilization

“…It is known that a transient increase in Ca2+ concentration occurs during fertilization in the sea urchin egg [Steinhardt et al, 1977;Epel, 1978;Eisen et al, 1984;Yoshimoto et al, 19851. We tested our hypothesis that this is the trigger for the accumulation of actin in the cortex.…”