Free calcium increases explosively in activating medaka eggs.

Abstract

We have used the calcium-specific lightemitting protein aequorin to follow changes in free calcium concentration during fertilization and cleavage of eggs from medaka, a fresh-water fish. Aequorin-injected meda a eggs show a very low resting glow before they are fertilized, indicating a low calcium concentration in the resting state. Upon activation by sperm, the calcium-mediated light emission increases to a level some 10,000 times the resting level with a 1 to 2 sec time constant for an e-fold increase, and then slowly returns to the resting level. Upon activation by the ionophore A23187, the early rise in luminescence is much slower, but once a threshold has been reached the subsequent rise becomes as rapid as the normal sperm-induced response. We infer that the explosive rise in calcium involves calcium-stimulated calcium release, and that a sperm normally triggers this rise by somehow inducing-a more modest and localized rise in calcium.One of the most important aspects of fertilization is the activation of the previously resting egg by the entering sperm. This normal activation response of the egg is dramatic and selfsustaining, and has long been thought to involve and require an increase of free calcium in the cytoplasm. Early workers conceived of a calcium theory of activation because a variety of eggs can be activated parthenogenetically (i.e., without sperm) simply by exposure to calcium-rich solutions, or by various disturbances, e.g., pricking the egg, in (and only in) a calcium-bearing medium (ref. 1; also compare refs. 2-5). More recently, the calcium theory has been supported by experiments showing that the calcium-specific ionophore A23187 can activate eggs (6-9).The results of previous efforts to measure a change in free calcium during activation have surely been consistent with a large increase, but are rather easily interpretable in other ways (2,8,10,11). For example, the 10-to 100-fold increase in 45Ca efflux during activation of sea urchin eggs (7, 11) might well be due to an increase in free cytoplasmic calcium, but might also represent the release of calcium from cortical vesicles.In this paper, we demonstrate a dramatic rise in free calcium concentration within the cytoplasm of activating medaka eggs by injecting them with the calcium-specific photoprotein aequorin. This calcium indicator has been previously used to detect calcium transients in muscle (12) and nerve (13), and is presently, we believe, the most sensitive and reliable method for detecting changes in free calcium within cells. The egg we have used is from the medaka, a fresh-water fish. The advantages of this~egg are: first, it is large (1.2 mm diameter); second, it can be injected without blocking subsequent fertilization (3); and third, it is transparent. A measurement was first made of the egg's "resting glow." Then the photomultiplier was turned off, the chamber was opened, sperm were added about 5 mm away from the egg, the chamber was closed, and the tube was turned back on. The calcium-mediated light emission ...

A free calcium wave traverses the activating egg of the medaka, Oryzias latipes.

“…It should be emphasized that the luminescent reaction (between aequorin and free calcium within the egg) is so slow that very little of the aequorin is used up during activation of the egg (43). Hence the fall in luminescence seen after the peak of light (in our previous study) and behind the moving zone of light (in this one) indicates a fall in free calcium rather than a fall in aequorin.…”

“…Specifically, two x 10 objectives were used: a Zeiss with a numerical aperture of 0.22 and a Leitz with one of 0.30. For comparison with previous results with a lensless system and a simple photomultiplier (43), it is important to realize that although the simple photomultiplier and the image intensifier should have comparable quantum efficieneies, the use of a lens radically reduces the quantum efficiency of the system as a whole. Specifically, one can estimate that only about 1% of the light emitted by an egg reaches the image intensifier through the above lenses: so the net quantum efficiency has been reduced about 100-fold in exchange for spatial information.…”

“…Unfertilized medaka eggs were obtained and injected with aequorin as described previously (43). It should be emphasized that the luminescent reaction (between aequorin and free calcium within the egg) is so slow that very little of the aequorin is used up during activation of the egg (43).…”

“…The first direct and convincing evidence of point one-a natural rise in free calcium-was recently reported for the egg of the medaka (Oryzias latipes), a fresh water oriental killifish widely used in student laboratories (43). This investigation utilized the calcium-specific photoprotein, aequorin, a remarkable substance which luminesces at a rate that increases with the level of free calcium.…”

Observations on intracellular pH during cleavage of eggs of Xenopus laevis.

“…Efforts to detect increases in the Ca" level during cleavage with the Ca-dependent photoprotein aequorin have been inconclusive in frog and fish eggs (1,21) and completely unsuccessful in the sea urchin egg (14) . A second investigation of Ca 21 in the frog egg with a Ca-sensitive microelectrode also found no changes in Ca`levels (22).…”