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Ca2+ hypothesis
- Calcium Signals for Egg Activation in Mammals

Ca2+ hypothesis

The concept of Ca2+ as a signal for egg activation arose in the early 1930s from experiments inducing artificial activation. The Ca2+ hypothesis, however, was substantiated 40 years later. In 1974, Steinhardt et al. showed that the Ca2+ ionophore A23187 activated sea urchin, starfish, toad, and hamster eggs (3). In 1977, an explosive [Ca2+]i rise was first recorded at fertilization in eggs of medaka fish and sea urchins using the Ca2+- binding luminescent protein aequorin (2). Furthermore, egg activation was induced by injection of Ca2+ buffer into the egg, and it was prevented when the [Ca2+]i rise was blocked by pre-injection of the Ca2+-chelating agent EGTA. Thus, the [Ca2+]i rise was considered to be the necessary and sufficient factor for egg activation, being the initial step of the post-fertilization cascade leading to early embryonic development. Later extensive research has proved that Ca2+-dependent egg activation is universal in the animal kingdom (1, 2).

Ca2+ wave

In 1978, Gilkey et al. (4) first displayed images of a Ca2+ wave that starts from the sperm fusion site and travels across the medaka egg (diameter, approximately 1 mm) at the velocity of approximately 10 μm/ s consistent with that of a wave of cortical granule exocytosis. The Ca2+ wave was not affected by external Ca2+ (4), indicating that the [Ca2+]i rise is due to intracellular Ca2+ release. The propagating nature was thought to be mediated by a positive feedback system based on “Ca2+- induced Ca2+ release” (CICR) found in the sarcoplasmic reticulum (SR) of muscle cells at that time (5). Later experiments have demonstrated that eggs of deuterostome animals (echinodermata such as sea urchin and starfish and chordata such as ascidia and vertebrates) exhibit Ca2+ waves due to intracellular Ca2+ release (Fig. 1), while eggs of protostome animals (from nemartia to annelida) show a non-wave-like, synchronous [Ca2+]i rise due to Ca2+ influx from outside the cell (1). In some protostome animals, however, multiple [Ca2+]i rises follow the first Ca2+ transient. Those Ca2+ oscillations are associated with waves caused by Ca2+ release from the ER (1, 2).

As to the mechanism of Ca2+ release from the ER, the inositol 1,4,5-trisphosphate receptor (IP3R)/Ca2+ channel is involved in all species that show Ca2+ waves (1, 2), determined by its inhibitor heparin or, more specifically, by a monoclonal antibody 18A10 against type 1 IP3R in mammalian eggs (6). Thus, IP3-induced Ca2+ release (IICR) plays an essential role in the Ca2+ response at fertilization (6) (Fig. 1), although the ryanodine receptor (RyR)/Ca2+ channel co-exists with the IP3R in sea urchin, mouse, and human eggs (1, 2). CICR is known as a characteristic of the RyR (7), but it can be mediated by the IP3R as well (Fig. 1), since Ca2+ itself is an activator of the IP3R (8), and the rate of IICR is enhanced by Ca2+ at the concentration between 100 nM ([Ca2+]i at the resting state of cells) and 300 nM in smooth muscle cells (7). In practice, it was demonstrated that a Ca2+ wave was induced in an all-or-none manner by injection of Ca2+ to the cortical cytoplasm of the golden hamster egg which lacks the RyR (6).

Ca2+ oscillations

In 1981, Miyazaki and Igusa (9) showed that a series of periodic hyperpolarizations occur at fertilization of hamster eggs. This suggested that repetitive [Ca2+]i rises likely accompany mammalian fertilization, as each hyperpolarization was due to Ca2+-activated K+ conductance increase. In 1986, [Ca2+]i rises were directly recorded by a Ca2+-sensitive microelectrode and by aequorin luminescence, and repetitive Ca2+ waves were displayed using a super-sensitive camera system (6). As more data accumulated, Ca2+ oscillations turned out to be common to mammalian eggs (6) and were found to occur in ascidian eggs and some protosome eggs (1). The first Ca2+ wave starts from the sperm fusion site, but later Ca2+ waves begin from the vegetal hemisphere irrespective of the sperm fusion site or, in ascidian eggs, from a certain pacemaker region rich in the ER near the vegetal pole (1, 2).

The rate of IICR is suppressed by increased [Ca2+]i over 300 nM, while enhanced by 100 – 300 nM [Ca2+]i (7). This bell-shaped dependence of IICR on [Ca2+]i is likely to be the basis for repetitive transient Ca2+ release from the ER. Ca2+ oscillations take place in mouse eggs in the presence of a non-metabolizable agonist of the IP3R, adenophostin B (2). The occurrence of frequent Ca2+ oscillations following a constant supply of IP3 can be explained by a single Ca2+ pool model based on IICR and Ca2+ influx (store-operated Ca2+ entry) (6). In mouse eggs, the first Ca2+ transient occurs a few min after sperm-egg fusion and lasts for several minutes (10). Subsequently, discrete Ca2+ spikes occur at intervals of approximately 10 min (Fig. 2A) (10). Meanwhile, the second polar body is formed as a result of the second meiotic division. The interval between Ca2+ spikes becomes longer up to 20 – 30 min. Each Ca2+ spike is generated when a preceding slow [Ca2+]i rise reaches a certain level. These Ca2+ response patterns are mimicked by slightly elevating the cytoplasmic IP3 level by release of caged IP3 (11). Thus, the spatiotemporal Ca2+ signal at fertilization can be produced by IP3R-mediated Ca2+ release. Ca2+ oscillations last for 3 – 5 h and cease when the 1-cell embryo enters the interphase of a cell cycle and forms the male and female pronuclei (Fig. 2A) (10, 11). Thus, Ca2+ oscillations in mouse eggs are cell cycle-dependent (see later section).

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