[phpBB Debug] PHP Warning: in file /home/biologyonline/public_html/kb/print.php on line 19: include(../common.php): failed to open stream: No such file or directory
[phpBB Debug] PHP Warning: in file /home/biologyonline/public_html/kb/print.php on line 19: include(): Failed opening '../common.php' for inclusion (include_path='.:/usr/share/pear:/usr/share/php')
Print - Biology-Online

Calcium Signals for Egg Activation in Mammals



Calcium Signals for Egg Activation in Mammals

Shunichi Miyazaki1,* and Masahiko Ito1

1Department of Physiology, Tokyo Women’s Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan

Received February 21, 2006

Abstract. A dramatic increase in intracellular Ca2+ concentration ([Ca2+i) occurs in eggs at fertilization common to all animal species examined to date, and this serves as a pivotal signal for egg activation characterized by resumption of meiotic cell division and formation of the pronuclei. In mammalian eggs, repetitive [Ca2+]i rises (Ca2+ oscillations) each of which accompanies a propagating wave across the egg occur due to release of Ca2+ from the endoplasmic reticulum mainly through type 1 inositol 1,4,5-trisphosphate (IP3) receptor. Ca2+ oscillations are induced by a cytosolic sperm factor driven into the egg cytoplasm upon spermegg fusion. A current strong candidate of the sperm factor is a novel sperm-specific isozyme of phospholipase C (IP3-producing enzyme), PLCζ. Recent extensive research has reveled characteristics of PLCζ such as the Ca2+ oscillation-inducing activity after injection of PLCζ- encoding RNA or recombinant PLCζ into mouse eggs, extremely high Ca2+-sensitivity of the enzymatic activity in vitro, and nuclear translocation ability possibly related to cell-cycledependent regulation of Ca2+ oscillations. [Ca2+]i rises cause successive activation of calmodulindependent kinase II and E3 ubiquitin ligase, lead to proteolysis of ubiquitinated cyclin B1 and inactivation of metaphase-promoting factor (Cdk1 /cyclin B1 complex), and result in the release of eggs from meiotic arrest.

Keywords: intracellular Ca2+, fertilization, mammalian egg, sperm factor, egg activation

Source: J Pharmacol Sci 100, 545 – 552 (2006).


Intracellular calcium ion is a key second messenger that regulates a wide variety of cellular functions. Egg activation at fertilization is one of the important Ca2+- dependent biological phenomena. In fertilization, the sperm not only provides one half of the genomes to the egg but also awakes the egg that is arrested at a certain stage of meiotic cell division in a species-specific manner. The release from the meiotic arrest is referred to as “egg activation” characterized by formation of the polar body and male and female pronuclei. Egg activation is caused by a dramatic increase in intracellular Ca2+ concentration ([Ca2+]i) common to every animal species ever examined (1, 2). The [Ca2+]i rise is mainly due to Ca2+ release from the endoplasmic reticulum (ER) and forms a “Ca2+ wave” that starts from the site of sperm-egg fusion and propagates the Ca2+ signal over the whole egg. Besides the spatial Ca2+ signal, repetitive [Ca2+]i rises designated as “Ca2+ oscillations” occur as temporal Ca2+ signals in various species including mammals (1, 2). In regard to the cell signaling in fertilization, there are two essential subjects upstream and downstream of the [Ca2+]i rise: how the sperm induces the [Ca2+]i rise and how the [Ca2+]i rise leads to egg activation. Here we review advances in the research on Ca2+ signals at fertilization with special attention to mammals.

Ca2+ hypothesis

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).

Sperm factor hypothesis

The signaling pathway leading to IICR at fertilization has been a central subject since the mid-80s. IP3 is produced by hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate (PIP2) by the aid of phospholipase C (PLC), referred to as the PI pathway. The receptor/G protein /PI pathway or receptor/protein tyrosine kinase (PTK)/PI pathway is well known in a wide variety of somatic cells. Although there are surface proteins that mediate sperm-egg binding (Fig. 1), no evidence for their link to intracellular signaling has been obtained (12). In 1990, Swann (13) showed that injection of hamster sperm extract into an egg is capable of inducing Ca2+ oscillations similar to those seen at fertilization. In mouse eggs, Ca2+ spikes induced by hamster sperm extract have higher frequency than those at fertilization (Fig. 2B). Although there was some difference in the Ca2+ spike frequency, the finding lead to the sperm factor hypothesis that predicts a cytosolic sperm factor, Ca2+ oscillation-inducing protein (COIP), is introduced into the egg cytoplasm upon sperm-egg fusion (Fig. 1). The predicted COIP is very important because it implies the existence of the sperm-derived egg-activation factor. The idea was reinforced by the following results (11, 12): Ca2+ oscillations are induced in human eggs upon intracytoplasmic (single) sperm injection (14), ICSI, which is a current powerful therapy of sterility. Namely, fertilization is possible without any sperm-egg surface interaction, if an assisted reproduction technology is used. Upon in vitro fertilization by usual insemination, Ca2+ oscillations in mouse eggs begin a few minutes ‘after’ sperm-egg fusion takes place. Ca2+ oscillations are completely prevented in CD9-knockout mouse eggs in which sperm-egg fusion is defective while sperm-egg binding still occurs (15). Thus, cytoplasmic sperm-egg continuity is prerequisite for inducing the Ca2+ response at fertilization (Fig. 1).

Ca2+ oscillation-inducing sperm protein

Studies have focused on the identification of COIP since 1990 by isolation and purification from sperm extract, associated with bioassay of Ca2+ oscillationinducing activity by injection of a sperm protein fraction into eggs. Several groups including us have been engaged in this work for 10 years, but no decisive candidate of COIP has been identified by this approach (2). Another approach is based upon the supposition that any messenger system related to the PI pathway is likely involved. PLCs are prime candidates for COIP. Actually, PLCβ1, γ1, γ2, δ1, and δ4 are known to be expressed in the mammalian sperm (16). However, recombinant PLCβ1, γ1, γ2, and δ1 all failed to cause Ca2+ release in the egg cytoplasm or caused Ca2+ release only at extremely high doses. In 2002, Saunders et al. identified a novel isozyme of PLC, PLC-zeta, that is specifically expressed in the mouse sperm (17). They also presented that injection of cRNA encoding PLCζ into mouse eggs can produce fertilization-like Ca2+ oscillations and subsequent early embryonic development up to the balatocyst and that the expressed level of PLCζ for initiation of Ca2+ oscillations was comparable to the amount estimated to be contained in a single mouse sperm. Furthermore, the Ca2+ oscillationinducing activity of sperm extract is lost when PLCζ is immunodepleted from the sperm extract. Thus, PLCζ has received much attention as the putative COIP (16, 17). Ca2+ oscillation-inducing activity of PLCζ Recently, characteristics of PLCζ has been extensively examined in relation to a candidate of the sperm factor. PLCζ is the smallest PLC isozyme identified to date; it is composed of four EF-hand domains in the N-terminus, X and Y catalytic domains, and C2 domain in the C-terminus (Fig. 3A) common to other PLC isozymes, but lacks a N-terminal pleckstrin homology (PH) domain (17). We expressed PLCζ in mouse eggs by injection of cRNA encoding PLCζ fused with a fluorescent protein ‘Venus’ which enabled us to monitor the expression level and distribution of PLCζ in the egg (18). Fertilization-like Ca2+ oscillations appear at 30 – 40 min after RNA injection (Fig. 3B), when expressed PLCζ reached 10 – 40 × 10−15 g in the egg, comparable to the estimated content in a single sperm (18). The frequency of Ca2+ spikes is progressively increased (Fig. 3B) because PLCζ is continuously expressed in this experiment. The egg is activated by PLCζ-mediated Ca2+ oscillations and forms the (female) pronucleus (PN).

Kouchi et al. (19) succeeded in synthesizing functional PLCζ using baculovirus/Sf9-cell expression system. Microinjection of recombinant PLCζ protein into mouse eggs induced serial Ca2+ spikes (Fig. 2C) quite similar to those produced by injection of sperm extract (Fig. 2B). PLCζ is similar to PLCδ1 (38% identity and 49% similarity in 647 amino acid residues of PLCζ), although the PH domain is present in PLCδ1 but absent in PLCζ. Recombinant PLCδ1 induced Ca2+ oscillations as well, but 20-fold higher concentration was required, compared with PLCζ (19). Since PLCζ as well as PLCδ lacks a regulatory domain such as the G protein-binding site of PLCβ or the SH domain of PLCγ for phosphorylation by PTK, the activation mechanism of PLCζ and PLCδ is unknown. It is necessary to access how PLCζ undergoes the active state for production of IP3. It is generally known that the enzymatic activity of PLC is enhanced by an increase in [Ca2+]. In vitro assay of PIP2-hydrolyzing activity at various [Ca2+] (plotting the specific activity in terms of percentage to the maximal activity) revealed that recombinant PLCζ has a significant activity at [Ca2+] as low as 10 nM and 70% maximal activity at 100 nM [Ca2+] (19) that is usually the basal [Ca2+]i level of cells. PLCζ activity is maximal at 1 μM [Ca2+], which is the peak [Ca2+]i level of each Ca2+ spike during Ca2+ oscillations in fertilized mouse eggs. EC50 was 52 nM for PLCζ, whereas it was 5.7 μM for PLCδ1; PLCζ has approximately 100-fold higher Ca2+-sensitivity. Such high Ca2+-sensitivity of PIP2-hydrolyzing activity of PLCζ is an appropriate characteristic as the sperm factor that is driven into the egg at fertilization, first triggers Ca2+ release without any preceding [Ca2+]i rise, and maintains long-lasting Ca2+ oscillations after fertilization.

Structure-function analysis of PLC-zeta

Structure-function analysis of PLCζ

A short form of PLCζ, s-PLCζ that lacks three EF-hand domains from the N-terminus is expressed in the mouse sperm, probably as a splicing variant. s-PLCζ has much less Ca2+ oscillation-inducing activity, compared with PLCζ: injection of RNA at 100-fold higher concentration barely induced Ca2+ oscillations after a delay of 3 h (18). This suggests that EF-hand domains which usually contain the Ca2+-binding site in a protein may be important for the enzymatic activity of PLCζ. Our molecular structure-function analysis showed that when 4 EF-hand domains (EF1 to EF4) from the Nterminus are deleted one-by-one, deletion of EF1 and EF2 of recombinant PLCζ causes marked reduction of the PIP2-hydrolyzing activity in vitro and loss of the Ca2+ oscillation-inducing activity in mouse eggs after injection of RNA encoding the mutant PLCζ (20).

However, deletion of EF1 and EF2 or mutation of EF1 or EF2 at the x and z positions of the putative Ca2+-binding loop little affects Ca2+-sensitivity of the PLC activity, while deletion of EF1 to EF3 caused 12-fold elevation of EC50 of Ca2+ concentration (20). Thus, EF1 and EF2 are important for the PLCζ activity, and EF3 is responsible for its high Ca2+ sensitivity. Deletion of EF1-4 or Cterminal C2 domain (Fig. 3A) results in complete loss of PLC activity (20), indicating that both regions are prerequisite for the PLCζ activity.

It seems that PLC activity of PLCζ may be derived from the highly coordinated structure of the EF-hand region and C2 domain rather than the primary sequence in the Ca2+-binding loop. According to crystal analysis of three dimensional structure, PLCδ1 is folded at the linker region between the X and Y catalytic domains in such a way that the C2 domain in the C-terminus makes extensive contact with EF-hand domains in the Nterminus and the catalytic domain, forming the catalytic core (21, 22). Considering PLCζ in analogy with PLCδ1, all domains have to be prepared to form the three dimensional active conformation. There may be Ca2+- dependent coordinating structural determinant(s) other than EF1-EF3 for the highly Ca2+-sensitive enzymatic activity.

C2 domain is known to play a significant role in Ca2+- dependent subcellular membrane targeting of several lipid-metabolizing enzymes such as PLCδ1 or cPLA2 (22). Screening of interaction between C2 domain and phosphoinositides has revealed that C2 has substantial affinity to PI(3)P and, to the lesser extent, to PI(5)P, but not to PI(4,5)P2 (20). Interestingly, these phosphatidylinositol monophosphates interfers with the PI(4,5)P2-hydrolyzing activity of PLCζ in vitro (20). The interaction between C2 domain and PI(3)P may be significant for negative regulation of PLCζ.

Nuclear translocation ability of PLC-zeta

Nuclear translocation ability of PLCζ

Another characteristic feature of PLCζ is the nuclear translocation ability. PLCζ-Venus expressed by injection of cRNA into mouse eggs is increased up to 3 h and attained a steady level at 4 – 5 h (18) (Fig. 3C). Expressed PLCζ distributes homogenously in the egg cytoplasm without special localization or membrane association. Interestingly, expressed PLCζ is accumulated into the PN formed at 5 – 6 h and continuously increased there (18) (Fig. 3C). This finding is consistent with earlier observation that the PN formed after fertilization has Ca2+ oscillation-inducing activity when introduced into a new unfertilized egg (23). Ca2+ oscillations after fertilization (10, 11) or after injection of PLCζ- encoding RNA (24) cease at about the time when the PN is formed (Fig. 2A), suggesting that sequestration of the sperm factor COIP may be the cause of the termination of Ca2+ oscillations. This idea is supported by the finding that Ca2+ oscillations do not stop but continue over 10 h when PN formation is prevented by injection of a lectin, WGA (25), or a point mutation is added to a putative nuclear translocation signal region of PLCζ (24). During early development, Ca2+ oscillations reappear in the 1-cell mouse embryo at the stage of nuclear envelope breakdown just before the first cell division (15 – 16 h after fertilization) and cease after the first cleavage (24) (Fig. 4). Correspondingly, when RNA encoding PLCζ-Venus is injected into the 1-cell embryo 6 h after fertilization in which the formed male and female pronuclei are visible, expressed PLCζ-Venus translolcates into the PN, and then accumulated PLCζ is liberated from the PN into the cytoplasm upon nuclear envelope breakdown (26) (Fig. 4). Subsequently, PLCζ- Venus translocates again into the nuclei of the 2-cell embryo after the first cleavage (24, 26) (Fig. 4). These results lead to the view that shuttling movement of PLCζ between the cytoplasm and nucleus may turn on and off a series of IP3-dependent [Ca2+]i rises in a cell cycle dependent manner.

Perspective of PLC-zeta

Perspective of PLCζ

The PLCζ gene has been cloned in mouse, rat, dog, pig, cow, monkey, and human. The Ca2+ oscillationinducing activity of sperm extract is compatible among mammalian species, although the grade of the activity seems distinct. Besides soluble cytosolic sperm factor, an insoluble component bound to the perinuclear matrix of the sperm has the egg-activating activity. A recent assay of the Ca2+ oscillation-inducing activity coupled to tandem mass spectrometry has shown that the activity of the component from the perinuclear matrix corresponded to PLCζ (27). It is important to elucidate in the near future whether PLCζ is the COIP that operates during physiological fertilization of mammalian eggs. The experiment using transgenic RNA interference of PLCζ has shown both reduction of the PLCζ content in spermatozoa of the transgenic mice and reduction of the number of Ca2+ spikes in eggs inseminated with the sperm (28). Since the number of Ca2+ spikes tends to be affected by experimental conditions of in vitro fertilization, it is desirable to show complete block of Ca2+ oscillations at fertilization by knocking out the PLCζ gene.

It is also interesting to examine the sperm factor from the phylogenic standpoint. As to fertilization signaling in non-mammalian eggs, the activation of PLCγ via Src-family PTK is involved at fertilization of sea urchin, starfish, ascidia, and frog (12) because Ca2+ responses at fertilization are prevented by pre-injection of the SH2 domain of PTK or PLCγ on the basis of a dominantnegative experiment. The mode of stimulation of the sperm is still unknown in these species. At present, evidence for the sperm factor hypothesis is accumulated in ascidia (2). Further studies are also necessary to examine the activation and modification mechanism of PLCζ on the basis of the molecular structure because PLCζ might be inactivated in the sperm to prevent [Ca2+]i rises before it is introduced into the egg cytoplasm. In this context, PLCζ bound to the perinuclear matrix might be important. PLCζ driven into the egg cytoplasm might require any aid of egg factor(s); for example, the accession to the membrane might be promoted by an egg factor, since PLCζ lacks the PH domain.

The nuclear translocation of PLCζ might have biological significance other than regulation of Ca2+ oscillations. The PI pathway exists in the nucleus, and PLC plays biological roles in the nucleus (29). For example, PLCβ1 translocates into the nucleus during G2/M transition in immature mouse oocytes and participates in germinal vesicle breakdown (30). It is predicted that diacylglycerol produced by PLCβ1 attracts PKCβII from the cytoplasm and PKCβII causes lamin phosphorylation leading to nuclear-envelope breakdown (29). PLCδ4 is predominantly present in the nucleus and increases dramatically at G1/ S transition in response to mitogenic stimulation (31). It is interesting to address whether PLCζ accumulated in the PN affects cell proliferation and differentiation in early embryonic development. PLCζ may be applied for artificial egg activation for stockbreeding. PLCζ could be utilized as an assisted reproduction technology in clinical medicine.

Signaling from the [Ca2+]i rise to egg activation

Signaling from the [Ca2+]i rise to egg activation

The signaling downstream the [Ca2+]i rise leading to egg activation is another essential subject. An increase in [Ca2+]i induces cortical granule exocytosis in various species (Fig. 4), as is generally the case in secretory cells. This event causes formation of the fertilization membrane and establishment of the block to polyspermy in sea urchin eggs. In mammals, the loss of cortical granules due to exocytotic secretion advances depending on the number of Ca2+ spikes (32). The released substance modifies proteins in the zona pellucida that surrounds the egg (zona reaction), resulting in polyspermy block (33).

[Ca2+]I rise triggers resumption of meiotic cell division. The second polar body is formed and subsequently the male and female pronuclei are formed (33) (Fig. 4). One of the central molecules in egg activation is maturation- (or metaphase-) promoting factor (MPF: Cdk1/cyclin B1 complex), a ubiquitous cell cycle regulator (34) (Fig. 4). Vertebrate mature eggs are arrested at the metaphase of the second meiosis (M II) due to sustained activity of MPF by the aid of cytostatic factor (CSF). Eggs are released from the M II arrest by fertilization as a consequence of the degradation of cyclin B1 by ubiqutin/proteasome-mediated proteolysis (Fig. 4). The Ca2+ response at fertilization is linked to activation of ubiqutin /proteasome. Upon [Ca2+]I rise at fertilization, Ca2+ binds to calmodulin and thereby activates calmodulin-dependent kinase II (CaMK II) (35). In fertilized mouse eggs, activation of CaMK II occurs at each Ca2+ peak (36). To be subjected to proteolysis, cyclin B1 is poly-ubiquitinated by anaphase promoting complex or cyclosome (APC/C), an E3 ubiquitin ligase,. This process is prevented by CSF (34, 37) (Fig. 4). In mammals, CaMK II causes inhibition of CSF, plausibly acting on the putative CSF component Emi 1, Mad2, or Bub1 (the most downstream component of the c-mos-MAPK (mitogen-activated protein kinase) pathway) (37). Taken together, the current view for the possible signaling pathway in mammals can be illustrated as in Fig. 4.

Repetitive [Ca2+]i rises at fertilization is necessary to accomplish degradation of MPF. If the number of Ca2+ spikes is insufficient, fertilized eggs are arrested at the M III stage after formation of the second polar body and fail to form the PN (32). Long-lasting Ca2+ oscillations in mouse eggs are responsible for pronucleus formation. Sufficient number of Ca2+ spikes causes reduction of MAPK activity and thereby leads to pronucleus formation (32) (Fig. 4). It has also been reported that the frequency and amplitude of Ca2+ oscillations affect the processes occurring much later during embryonic development in rabbits such as compaction, blastocyst formation, and the rate of successful transplantation of 4-cell embryo to host mothers (13).

The mechanism of fertilization has not been fully revealed, despite the long research history over a century. The sperm factor /Ca2+ increase in the egg /egg activation described here is the central point of cell signaling at fertilization, and research on this point is the most advanced in mammals at present. Further understanding of the mechanism involved in fertilization and early embryonic development depends on future studies.


This work was supported by Grant-in-Aid for General Scientific Research (B) to S.M. from the Japan Ministry of Education, Culture, Sports, Science, and Technology.


1 Stricker SA. Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev Biol. 1999; 211:157–176.

2 Miyazaki S. Thirty years of calcium signals at fertilization. Semin Cell Dev Biol. In prerss 2006.

3 Steinhardt RA, Epel D, Carroll EJ Jr, Yanagimachi R. Is calcium ionophore a universal activator for unfertilized eggs? Nature. 1974;252:41–43.

4 Gilkey JC, Jaffe LF, Ridgeway EB, Reynolds GT. A free calcium wave traverses the activating eggs of the medaka, Oryzias latipes. J Cell Biol. 1978;76:448–466.

5 Endo M. Calcium release from the sarcoplasmic reticulum. Physiol Rev. 1977;57:71–108.

6 Miyazaki S, Shirakawa H, Nakada K, Honda Y. Essential role of the inositol 1,4,5-trisphosphate receptor /Ca2+ release channel in Ca2+ waves and Ca2+ oscillations at fertilization of mammalian eggs. Dev Biol. 1993;158:62–78.

7 Iino M, Endo M. Calcium-dependent immediate feedback control of inositol 1,4,5-trisphosphate-induced Ca2+ release. Nature. 1992;360:76–78.

8 Furuichi T, Kohda K, Miyawaki A, Mikoshiba K. Intracellular channels. Curr Opin Cell Biol. 1994;4:294–303.

9 Miyazaki S, Igusa Y. Fertilization potential in golden hamster eggs consists of recurring hyperpolarizations Nature. 1981;290: 702–704.

10 Deguchi R, Shirakawa H, Oda S, Mohri T, Miyazaki S. Spatiotemporal analysis of Ca2+ waves in relation to the sperm entry site and animal-vegetal axis during Ca2+ oscillations in fertilized mouse eggs. Dev Biol. 2000;218:299–313.

11 Jones KT. Ca2+ oscillations in the activation of the egg and development of the embryo in mammals. Int J Dev Biol. 1998;42:1–10.

12 Runft LL, Jaffe LA, Mehlmann LM. Egg activation at fertilization: where it all begins. Dev Biol. 2002;245:237–254.

13 Swann K, Ozil J-P. Dynamics of the calcium signal that triggers mammalian egg activation. Int Rev Cytol. 1994;152:183–222.

14 Tesarik J, Sousa M, Testart J. Human oocyte activation after intracytoplasmic sperm injection. Human Reprod. 1994;9:511– 518.

15 Kaji K, Oda S, Shikano T, Ohnuki T, Uematsu Y, Sakagami J, et al. The gamete fusion process is defective in eggs of Cd9- deficient mice. Nature Genet. 2000;24:279–282.

16 Swann K, Larman MG, Saunders CM, Lai FA. The cytosolic sperm factor that triggers Ca2+ oscillations and egg activation in mammals is a novel phospholipase C: PLCζ. Reproduction. 2004;127:431–439.

17 Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, et al. PLCζ: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development. Development. 2002;129;3533–3544.

18 Yoda A, Oda S, Shikano T, Kouchi Z, Awaji T, Shirakawa H, et al. Ca2+ oscillation-inducing phospholipase C zeta expressed in mouse eggs is accumulated to the pronucleus during egg activation. Dev Biol. 2004;268:245–257.

19 Kouchi Z, Fukami K, Shikano T, Oda S, Nakamura Y, Takenawa T, et al. Recombinant phospholipase Cζ has high Ca2+ sensitivity and induces Ca2+ oscillations in mouse eggs. J Biol Chem. 2004;279:10408–10412.

20 Kouchi Z, Shikano T, Nakamura Y, Shirakawa H, Fukami K, Miyazaki S. The role of EF-hand domains and C2 domain in regulation of enzymatic activity of phospholipase Cζ. J Biol Chem. 2005;280:21015–21021.

21 Essen L-O, Perisic O, Cheung R, Katan M, Williams RL. Crystal structure of a mammalian phosphoinositide-specific phospholipase Cδ. Nature. 1996;380:595–602.

22 Williams RL. Mammalian phosphoinositide-specific phospholipase C. Biochim Biophys Acta. 1999;1441:255–267.

23 Kono T, Carroll J, Swann K, Whittingham DG. Nuclei from fertilized mouse embryos have calcium-releasing activity. Development. 1995;121:1123–1128.

24 Larman MG, Saunders CM, Carroll J, Lai FA, Swann K. Cell cycle-dependent Ca2+ oscillations in mouse embryos are regulated by nuclear targeting of PLCζ. J Cell Sci. 2004;117: 2513–2521.

25 Marangos P, FitzHarris G, Carroll J. Ca2+ oscillations at fertilization in mammalians are regulated by the formation of pronuclei. Development. 2003;130:1461–1472.

26 Sone Y, Ito M, Shirakawa H, Shikano T, Takeuchi H, Kinoshita K, et al. Nuclear translocation of phospholipase C-zeta, an eggactivating factor, during early embryonic development. Biochem Biophys Res Commun. 2005;330:690–694.

27 Fujimoto S, Yoshida N, Fukui T, Amanai M, Isobe T, Itagaki C, et al. Mammalian phospholipase Cζ induces oocyte activation from the sperm perinuclear matrix. Dev Biol. 2004;274:370– 383.

28 Knott JG, Kurokawa M, Fissore RA, Schultz RM, Williams CJ. Transgenic RNA interference reveals role for mouse sperm phospholipase Cζ in triggering Ca2+ oscillations during fertilization. Biol Reprod. 2005;72:992–996.

29 Irvine RF. Nuclear lipid signaling. Nat Rev Mol Cell Biol. 2003:4;349–360.

30 Avazeri N, Courtot AM, Pesty A, Duquenne C, Lefèvre B. Cytoplasmic and nuclear phospholipase C-β1 relocation: role in resumption of meiosis in the mouse oocyte. Mol Biol Cell. 2000;11:4369–4380.

31 Liu N, Fukami K, Yu H, Takenawa T. A new phospholipase C δ4 is induced at S-phase of the cell cycle and appears in the nucleus. J Biol Chem. 1996;271:355–360.

32 Ducibella T, Huneau D, Agelichio E, Xu Z, Schultz RM, Kopf GS, et al. Egg-to-embryo transition is driven by differential responses to Ca2+ oscillation number. Dev Biol. 2002;250:280– 291.

33 Yanagimachi R. Mammalian fertilization. In: Knobl E, Neill J, editors. Physiology of reproduction. New York: Raven Press; 1994. p. 189–317.

34 Jones KT. Turning it on and off: M-phase promoting factor during meiotic maturation and fertilization. Mol Hum Reprod. 2004;10:1–5.

35 Lorca T, Cruzalegui FH, Fesquet D, Cavadore JC, Mery J, Means A, et al. Calmodulin-dependent kinase II mediates inactivation of MPF and CSF upon fertilization of Xenopus eggs. Nature. 1993;366:270–273.

36 Markoulaki S, Matson S, Ducibella T. Fertilization stimulates long-lasting oscillations of CaMKII activity in mouse eggs. Dev Biol. 2004;272:15–25.

37 Hyslop LA, Nixon VL, Levasseur M, Chapman F, Chiba K, McDougall A. Ca2+-promoted cyclin B1 degradation in mouse oocytes requires the establishment of a metaphase arrest. Dev Biol. 2004;269:206–219.



Fig. 1. Schematic illustration of sperm-egg signaling that leads to successive Ca2+ release from the ER through the IP3R and form

figure 1


 Fig. 2. Ca2+ oscillations in mouse eggs induced by a spermatozoon at fertilization (A), injection of hamster sperm extract (B), and injection of recombinant PLC. (60 µg/ml) (C). The ordinate is the ratio of fluorescence intensity of fura-2 in the egg activated by 340 and 380 nm lights (F340/F380), reflecting [Ca2+]i.s a Ca2+ wave in mammalian eggs.sword,

figure 2


 Fig. 3. A: The domain feature of PLC. molecule. B: Ca2+ oscillations after injection of RNA (20 µg/ml) encoding PLC.-Venus. C: Distribution of expressed PLC.-Venus and translocation into the PN. Changes in the fluorescence intensity (arbitrary unit) of Venus in the cytoplasm and pronucleus are presented in 3 eggs. Inset is the bright field image and fluorescence image of a 1-cell embryo at 7.5 h after RNA injection and shows accumulation of expressed PLC. in the PN formed at 4 h. The arrow in the bright field image indicates the PN..

  figure 3



Fig. 4. Possible signaling pathway downstream the [Ca2+]i rise at fertilization leading to release from M II arrest, formation of the pronuclei, and 1st cleavage of mammalian embryos. The PN of 1-cell embryo and the nuclei of 2-cell embryo are shaded to illustrate the accumulation of the sperm factor or PLC..


figure 4