Intracellular calcium oscillations in mammalian eggs at fertilization

I am honoured to give a classical perspective on our old paper: Igusa & Miyazaki (1983).

In 1981, we first reported that a change in the membrane potential at fertilization, the fertilization potential, in golden hamster eggs consists of recurring hyperpolarizations (Miyazaki & Igusa, 1981), in contrast to the potential in sea urchin or starfish eggs, which is a depolarization involving an overshoot and lasting for ∼10 min. The in vitro fertilization technique in mammals was developed during the 1970s, but no physiological recording of fertilization events had been performed. Each hyperpolarizing response (HR) in fertilized hamster eggs began from the resting potential of −25 mV and reached −65 mV, having a total duration of 10 s. A series of HRs occurred every 50–60 s and lasted for several hours. Since each HR was found to be due to a Ca²⁺-activated K⁺ conductance increase, HRs indirectly indicated transient repetitive increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i). HRs had been reported in fibroblasts and sympathetic ganglion neurons, and were a good indicator of Ca²⁺ signals at a time when direct measurement of [Ca²⁺]_i was unavailable except for giant cells such as medaka fish eggs.

Our paper (Igusa & Miyazaki, 1983) suggested two significant areas for later study: for physiology, the linkage of Ca²⁺ influx from outside of the cell to intracellular Ca²⁺ release from Ca²⁺ stores for the generation of long-lasting repetitive [Ca²⁺]_i increase, which was later called intracellular Ca²⁺ oscillation, and for biology, Ca²⁺ oscillations that turned out to be the egg-activating signal characteristic of the fertilization of mammalian eggs. The paper showed that intervals between HRs are shortened by increasing external [Ca²⁺] or by hyperpolarization produced by passing continuous current through an intracellular microelectrode, i.e. by increasing the chemical or electrical driving force for Ca²⁺ influx, respectively. Intervals are prolonged by lowering external [Ca²⁺] or by depolarization, and HRs cease in Ca²⁺-free medium. HRs produced by injection of Ca²⁺ into the egg suggested that Ca²⁺-induced Ca²⁺ release would occur from Ca²⁺ stores when [Ca²⁺]_i reached a critical level. It was hypothesized that persistent Ca²⁺ entry plays a critical role in providing Ca²⁺ to refill Ca²⁺ stores and maintain a repetitive [Ca²⁺]_i rise due to repeated Ca²⁺ release.

In the middle of the 1980s, [Ca²⁺]_i measurement became possible in egg cells, which are larger than somatic cells. Repetitive [Ca²⁺]_i rise was recorded in hamster eggs using a Ca²⁺-sensitive microelectrode (Igusa & Miyazaki, 1986), and a Ca²⁺ wave was exhibited in each Ca²⁺ transient by a Ca²⁺-imaging method with the Ca²⁺-binding luminescent protein aequorin previously injected into the egg and using a super-sensitive camera system (Miyazaki et al. 1986). Ca²⁺ imaging was much more advanced around 1990 by utilizing Ca²⁺-binding fluorescent dyes such as fura-2 and by utilizing computers, and became applicable to somatic cells. It became a general concept that Ca²⁺ waves serve as a spatial Ca²⁺ signal propagating through the cell and Ca²⁺ oscillations are a temporal Ca²⁺ signal providing frequency-encoded cell signalling.

In 1987–88, the ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP₃R) were purified as Ca²⁺ release channels in the endoplasmic reticulum (ER). Ca²⁺ waves and Ca²⁺ oscillations in fertilized hamster eggs were shown to be due to Ca²⁺ release from the ER exclusively through IP₃R, and based on Ca²⁺-induced Ca²⁺ release mediated by IP₃R instead of RyR (Miyazaki et al. 1993). The idea proposed in 1983 was extended to a single IP₃-sensitive Ca²⁺ pool/Ca²⁺ entry model (Berridge & Dupont, 1994) with theoretical simulation (De Young & Keizer, 1992) for Ca²⁺ oscillations in somatic cells. The idea was substantiated by the discovery of capacitative (or store-operated) Ca²⁺ influx in various cells: Ca²⁺ entry that is coupled with Ca²⁺ release (Parekh & Penner, 1997). The store-operated Ca²⁺ influx pathway has been extensively analysed to date. As to Ca²⁺ dynamics underlying HRs, later experiment gave evidence utilizing Mn²⁺ quenching of intracellular fura-2 in mouse eggs that Mn²⁺ added to the extracellular medium enters the cytoplasm through the store-operated Ca²⁺ influx pathway, and is sequestered into the ER, released from the ER through IP₃R, and extruded to the exterior (Mohri et al. 2001).

A dramatic increase of [Ca²⁺]_i at fertilization has been recorded in a wide variety of eggs since the later half of the 1980s, and it is a pivotal signal for egg activation common to all species examined to date (see reviews by Stricker, 1999; Miyazaki, 2006). Unfertilized eggs are arrested at a certain stage of meiosis, and the [Ca²⁺]_i increase at fertilization induces release from the arrest, i.e. egg activation. Ca²⁺ oscillations consisting of transient Ca²⁺ spikes turned out to be the common and characteristic Ca²⁺ response in mammalian eggs. It has been shown in mouse eggs that the initial several Ca²⁺ spikes cause release from the metaphase of the second meiosis by activation of Ca²⁺- and calmodulin-dependent kinase II (CaMK II) leading to inactivation of the metaphase promoting factor (MPF) (see review by Ducibella et al. 2006). In mammalian eggs, the male and female pronuclei are formed 5 h or longer after sperm–egg fusion. Later Ca²⁺ spikes are responsible for pronucleus formation via reduction of mitogen-activated protein kinase (MAPK) activity (Ducibella et al. 2006). A series of Ca²⁺ spikes cease at about the time of pronucleus formation, at the interphase of the cell cycle. Thus, the generation of Ca²⁺ oscillations is cell cycle dependent.

It is of prime importantance to identify the sperm factor that induces Ca²⁺ oscillation in the egg, as it is the egg-activating factor. Evidence shows that the Ca²⁺ oscillation-inducing protein (COIP) is driven from the sperm cytoplasm into the ooplasm upon sperm–egg fusion, instead of sperm–egg surface receptor interaction leading to Ca²⁺ release from the ER (see review by Swann et al. 2006). The current strong candidate of COIP is a novel isozyme ‘zeta’ of phspholipase C (PLCζ) that hydrolyses membrane phosphatidylinositol 4,5-bisphosphate into IP₃ and diacylglycerol. PLCζ is specifically expressed in the mammalian sperm, and possesses appropriate characteristics to be the sperm factor that is introduced into the egg, first triggers Ca²⁺ release, and maintains Ca²⁺ oscillations (Swann et al. 2006; Miyazaki, 2006). At present, the study of the egg-activating sperm factor is the most advanced in mammals, compared with other kinds of animals. It remains to be elucidated that PLCζ actually functions as the sole sperm-derived egg-activating factor in physiological fertilization.

After our paper on repetitive HRs, unexpectedly extensive advance has come in the study of spatiotemporal Ca²⁺ dynamics involving Ca²⁺ release/Ca²⁺ influx coupling mechanism and Ca²⁺ oscillations as the key factor for egg activation and early embryonic development after fertilization of mammalian eggs.