The role of intracellular Ca²⁺ signaling during fertilization, as illustrated at the top of Fig. 6.1, is well established and has been extensively reviewed elsewhere (Wakai and Fissore 2013; Ducibella and Fissore 2008). Fusion of sperm and egg triggers a series of cellular events known collectively as egg activation , which transitions the fertilized egg into embryogenesis. Activation of the metaphase II-arrested egg induces cortical granule exocytosis, molecular remodeling of the zona pellucida and plasma membrane, translational activation of stored maternal mRNA, resumption and completion of meiosis, extrusion of the second polar body, and pronuclear formation (Schultz and Kopf 1995).

Sperm-egg fusion, or experimental injection of a sperm, rapidly initiates PLC-dependent production of IP3 and activation of the ITPR to generate a large transient increase in the concentration of cytosolic free Ca²⁺, followed by a series of low frequency Ca²⁺ oscillations (Fig. 6.1) that last for several hours (Miyazaki et al. 1993; Cuthbertson et al. 1981). The production of these Ca²⁺ oscillations requires not only the initial burst of IP3, but also Ca²⁺ reuptake by the ER, Ca²⁺ influx across the plasma membrane, regulation of ER Ca²⁺ channels and Ca²⁺ buffering by other organelles (Wakai and Fissore 2013). The oocyte contains most isoforms of PLC, many which are capable of being activated by ligand-receptor interactions during fusion of the sperm and egg. However, IP3 production in the fertilized egg is initiated by PLCζ (Fig. 6.1), an isoform not expressed by the oocyte, but delivered by the entering spermatozoon (Swann and Lai 2013; Saunders et al. 2002). The decondensing sperm head releases PLCζ into the ooplasm where, unlike other PLC isoforms that utilize plasma membrane PIP2, it appears to hydrolyze PIP2 associated with small (< 1 μm), cytoplasmic vesicles. Accumulation of IP3 in the vicinity of cortical ER near the sperm head produces localized release of Ca²⁺ that propagates a rapidly growing wave of cytosolic free Ca²⁺ across the egg during the initial transient, but not during later oscillations (Deguchi et al. 2000). The necessity of Ca²⁺ signaling at fertilization is underscored by a subgroup of infertile men who produce morphologically normal sperm that fail to properly express PLCζ and are, thus, unable to activate eggs, even when injected (Yoon et al. 2008; Kashir et al. 2010).

One effect of Ca²⁺ signaling could be a switch in cellular metabolism in preparation for the egg to embryo transition. Several key mitochondrial enzymes are activated by Ca²⁺ to increase the production of ATP through oxidative phosphorylation (Campbell and Swann 2006), providing energy to initiate the embryonic developmental program and activate SERCA for restoring Ca²⁺ to the ER at the completion of each Ca²⁺ oscillation (Dumollard et al. 2004; Liu et al. 2001; Wakai et al. 2013). With each rise of cytosolic Ca²⁺, the release of ATP to activate the SERCA pump would terminate the transient and replenish the ER Ca²⁺ stores. The progression of egg activation involves an extensive network of protein kinase reactions, cytoskeletal rearrangements and de novo protein biosynthesis, which each require an infusion of energy.

Ca²⁺ signaling rapidly induces the cortical reaction and resumption of meiosis through mechanisms dependent on Ca²⁺-binding proteins. During oocyte maturation, cortical granules redistribute from the cytoplasm to the oocyte cortex adjacent to the plasma membrane where they are poised to secrete their contents once Ca²⁺ becomes available (Ducibella 1996). Thus, the Ca²⁺ transient initiated by PLCζ triggers the cortical reaction, which provides multiple blocks to polyspermy through the release of proteins that modify the zona pellucida and plasma membrane. The events of oocyte activation are driven to a large extent by protein kinases, which are directly or indirectly activated downstream of Ca²⁺ (Ducibella and Fissore 2008). For example, calmodulin (CaM) is a Ca²⁺-binding protein that can activate a number of protein kinases, including myosin light chain kinase, which is an immediate effector of cortical granule exocytosis during Ca²⁺ oscillations (Ducibella and Fissore 2008). Another downstream target of CaM, Ca²⁺/calmodulin kinase II (CaMKII), initiates cell cycle resumption in fertilized eggs by phosphorylating Emi2 to lift inhibition of the cyclosome complex, which contains E3 ubiquitin ligase that destroys cyclin B1, thus, relieving the inhibition of meiosis by maturation promoting factor or MPF (Nixon et al. 2002; Madgwick et al. 2005). Protein kinase C (PKC), activated by Ca²⁺ and the PLC product, DAG, appears to contribute to multiple processes that drive the resumption of meiosis in eggs (Ducibella and Fissore 2008). Additionally, PKC activation and translocation to the plasma membrane occurs with each Ca²⁺ rise to trigger SOCE, which amplifies the oscillation (Halet et al. 2004).

By recruiting Ca²⁺-activated signaling pathways to produce the physiological changes that drive meiosis, DNA synthesis, mitosis and other activities associated with egg activation , Ca²⁺ provides a ubiquitous signal that sequentially initiates highly diverse cellular events during the egg to embryo transition at fertilization. It has been demonstrated by experimentally inducing Ca²⁺ transients in unfertilized mouse eggs that different numbers of Ca²⁺ oscillations (Fig. 6.1) are required to activate different events during egg activation (Ducibella et al. 2002). It was reported that cortical granule exocytosis increased with each Ca²⁺ oscillation, beginning at the first one, while cell cycle resumption required 4 oscillations to begin, polar body extrusion required 8 and pronuclear formation 24. At the molecular level, the decrease in histone H1 kinase and MAPK activities required for cell cycle resumption were not observed until after 8 and 24 oscillations, respectively. Synthesis of new proteins from stored maternal message was first clearly observed in eggs receiving 8 Ca²⁺ oscillations and increased further with additional transients. There was also evidence of a return to the initial molecular state in eggs receiving less than the full number of Ca²⁺ transients. It has been suggested that Ca²⁺ signaling sequentially induces cellular processes through accumulating downstream reactions mediated by Ca²⁺-dependent protein kinases and other Ca²⁺-binding proteins (Ducibella and Fissore 2008).

The spatiotemporal complexity of Ca²⁺ signaling at fertilization reflects the diversity of cellular processes that are orchestrated throughout the course of egg activation , and the adverse outcomes associated with deviations from the normal pattern of oscillations (Ducibella and Fissore 2008). The programming of the fertilized egg to generate a series of transient increases in the intracellular Ca²⁺ concentration (Dumollard et al. 2002) provides an internal molecular clock that directs sequentially enfolding events of egg activation (Ducibella and Fissore 2008). Clearly, signaling downstream of Ca²⁺ is a high-level process that can operate through Ca²⁺-binding proteins and downstream pathways in other dynamic developing systems. The remaining sections will present evidence that intracellular Ca²⁺ does indeed continue to provide a master signal that regulates preimplantation embryogenesis spatiotemporally, and translates signals from the female reproductive tract to coordinate maternal and embryonic development.

The Ca²⁺ oscillations induced at fertilization discontinue prior to first cleavage of the zygote (Cuthbertson et al. 1981), but the role of intracellular Ca²⁺ signaling does not end there. The success of preimplantation development depends on autocrine signaling by a number of trophic factors secreted from embryos . For this reason, embryos cultured as groups in small volumes promptly cleave, and produce blastocysts in greater number than embryos cultured singly in a large volume to dilute any accumulating secretions (Rappolee et al. 1988; Paria and Dey 1990; DeChiara et al. 1990; Palmieri et al. 1992; Schultz and Heyner 1992; Gardner et al. 1994; Wiley et al. 1995; O’Neill 1997). Embryos cultured in large volumes can, to some extent, be rescued by supplementing them with autocrine factors, including members of the epidermal growth factor (EGF) family and platelet-activating factor (PAF) , an ether phospholipid (1-o-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine). Autocrine signaling is most critical at the 2-cell stage and is not mitogenic in nature, but rather ensures cell survival by reducing apoptosis (Brison and Schultz 1997, 1998; O’Neill 1998). While, there are most likely a large number of secreted autocrine factors required for proper early preimplantation development, intracellular Ca²⁺ constitutes a key common pathway for many growth factors and cytokines .

PAF signaling during preimplantation development has been examined extensively by O’Neill and co-workers during the past two decades (O’Neill et al. 2012) and provides an example of the central role of intracellular Ca²⁺ signaling during the period immediately after fertilization. Mouse zygotes or 2-cell embryos respond to PAF through a pertussis-sensitive GPCR (Honda et al. 2002) that can activate Gαq/11, Gαi, Gαo and Gβγ to regulate a wide variety of downstream signaling pathways, depending on their availability. In 2-cell embryos, PAF activates phosphatidylinositol-3 kinase (PI3K), most likely through Gβγ, to produce phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 is a docking site for pleckstrin homology domains that in early mouse embryos activates both PLC and a voltage-gated Ca²⁺ channel. Both intracellular Ca²⁺ release from the ER governed by the ITPR and Ca²⁺ influx across the plasma membrane though L-type channels are required to elevate the cytoplasmic free Ca²⁺ (Lu et al. 2003; Emerson et al. 2000). The Ca²⁺ transient persists for 5 min before the channels become desensitized. The channels recover after 90 min to 2 h, producing periodic Ca²⁺ transients that promote survival, according to studies in which the sources of Ca²⁺ were disrupted with inhibitors, or by using embryos from mice lacking the PAF receptor gene (Lu et al. 2004).

Ca²⁺ transients generated by PAF (middle of Fig. 6.1) activate CaM and dependent kinases, including CaMKII, which results in elevated phosphorylation of the cAMP responsive element binding protein (CREB) that can homodimerize or heterodimerize with another transcription factor, ATF1, to induce new transcription (Jin and O’Neill 2010). The phosphorylation of CREB is associated with its nuclear localization. There is not an absolute requirement of PAF for CREB phosphorylation, but rather an enhancement of signaling that contributes significantly to the development of competent embryos with adequate numbers of pluripotent stem cells at the blastocyst stage. Presumably, PAF signaling downstream of Ca²⁺, CaM and their target proteins could be complex and awaits further investigation. It has been suggested that PAF activation of CREB could contribute to the activation of transcription from the embryonic genome, possibly as one of many newly recruited or activated transcription factors working in combination with constitutively expressed factors to initiate embryonic gene expression during the 2-cell stage of murine development (O’Neill et al. 2012). Nothing is currently known about the role of Ca²⁺ signaling in embryonic genome activation, but it is interesting to speculate that activated transcription factors accumulate with repetitive cycles of intracellular Ca²⁺ transients to regulate this crucial event temporally, beginning at fertilization and continuing into embryogenesis.

Pathways downstream of intracellular Ca²⁺ that advance formation and development of the morula are less well understood. Extracellular Ca²⁺ regulates compaction through its interaction with adhesion molecules on the surfaces of blastomeres (Ducibella and Anderson 1975), setting the stage for cavitation. Regions of cell-cell contact that form adherens junctions are occupied by E-cadherin, a homophilic binding protein that requires Ca²⁺ ions, and is functionally disrupted by their removal (Hirano et al. 1987). While extracellular Ca²⁺ is required during compaction for surface adhesion molecules, including E-cadherin (Ohsugi et al. 1997), there is also evidence that intracellular Ca²⁺ is required to maintain compaction (Pey et al. 1998). Compaction is highly dependent on the activity of the Ca²⁺-requiring, cytoplasmic enzyme PKC (Winkel et al. 1990; Ohsugi et al. 1993, 1994). An intracellular Ca²⁺ transient is generated when morulae are decompacted by removal of extracellular Ca²⁺ or inhibition of PKC, CaM, voltage-gated Ca²⁺ channels or microfilaments (Pey et al. 1998). These treatments do not alter intracellular Ca²⁺ concentrations prior to compaction, indicating a specific association with the compacted state. Additionally, Ca²⁺ signaling does not cause these disruptions within the embryo, as induction of a Ca²⁺ transient with ionomycin has no detrimental effect on compacted embryos. One interpretation of these findings is that Ca²⁺ is released from binding proteins into the cytoplasm by disrupting cell shape and adhesion of the morula, suggesting that maintenance of the compacted state is dependent on regulatory Ca²⁺-protein interactions.

A role for Ca²⁺ signaling throughout preimplantation development was first hypothesized from studies of the teratogen ethanol (Armant and Saunders 1996). Ethanol causes fetal alcohol spectrum disorder subsequent to maternal alcohol consumption during the post-implantation period, leading to central nervous system defects, craniofacial deformities and fetal growth restriction (Hannigan and Armant 2000). High concentrations of ethanol can be toxic to embryos during preimplantation development, but after exposure of mouse embryos at the 1-cell or 2-cell stage to relatively mild concentrations of approximately 0.1 % (~ 25 mM) ethanol, in vitro development to blastocyst significantly increases towards the in vivo rate, with increased cell numbers and accelerated differentiation of the trophoblast cells to a migratory phenotype (Leach et al. 1993). Considering the known ability of ethanol to activate mouse eggs by elevating intracellular Ca²⁺ (Cuthbertson et al. 1981) and the role of Ca²⁺ signaling downstream of embryotrophic factors such as PAF (Emerson et al. 2000), it is feasible that ethanol operates through a similar Ca²⁺-mediated mechanism. Evidence supporting a role for Ca²⁺ signaling in advancing the program for blastocyst formation has now accumulated from studies conducted mainly in the mouse model.

Mouse embryos exposed to ethanol during culture from the 8-cell stage cavitate faster than control embryos (Stachecki et al. 1994b). Exposure for 5 min is as effective as a 24-h exposure or a brief exposure to Ca²⁺ ionophore, suggesting a rapid Ca²⁺ signaling mechanism. Indeed, real time monitoring with the fluorescent intracellular Ca²⁺ indicator fluo-3-AM demonstrates a Ca²⁺ transient immediately after exposure to 0.1 % ethanol that is independent of extracellular Ca²⁺ and persists for about 5 min. Chelation of intracellular Ca²⁺ by pretreating embryos with BAPTA-AM prevents elevation of intracellular Ca²⁺ and attenuates the acceleration of cavitation rates when 8-cell embryos are exposed either to ethanol or the Ca²⁺ ionophore, ionomycin (Stachecki and Armant 1996b).

The source of cytoplasmic Ca²⁺ in cavitating preimplantation mouse embryos has been examined. Ca²⁺ transients induced at the 8-cell stage by ethanol are attenuated in embryos pretreated with inhibitors of PLC, and PLC inhibition significantly delays fluid accumulation during blastocyst formation (Stachecki and Armant 1996a). The latter finding together with the observed delay of basal cavitation rates by BAPTA-AM treatment (Stachecki and Armant 1996b) reveals an explicit requirement for Ca²⁺ and IP3 signaling during this phase of embryonic development. Activation of the ITPR with thimerosal induces a Ca²⁺ transient in embryos, but the RYR agonist caffeine is without affect (Stachecki and Armant 1996b), consistent with a critical role for the ITPR. Treatment of semipermeabilized embryos with IP3, but not ryanodine, initiates a large Ca²⁺ transient that is blocked by BAPTA-AM pretreatment, demonstrating robust mobilization of Ca²⁺ stores through the IP3 pathway (Stachecki and Armant 1996b). Similar experiments have been conducted by microinjection of IP3 or ryanodine into mouse eggs with comparable results (Kline and Kline 1994), suggesting that this mechanism is retained from an earlier stage, although the extrinsic triggers responsible might differ. The concentration of intracellular Ca²⁺ and the rate of blastocoel formation were not increased by adding DAG, the other product of PLC enzymatic activity, nor did PKC inhibitors attenuate the acceleration of cavitation by ethanol, demonstrating that IP3 and Ca²⁺ are exclusively responsible for transmitting the observed stimulatory effects (Stachecki and Armant 1996a). Whereas PAF could provide the stimulus in zygotes and 2-cell embryos (Emerson et al. 2000), lysophosphatidic acid (LPA; Fig. 6.1, bottom left), another bioactive phospholipid produced in the uterus (Tokumura et al. 2000), generates PLC-dependent Ca²⁺ transients in 8-cell mouse embryos (Stachecki and Armant 1996a). Production of these and other embryotrophic factors by embryos and the reproductive tract could provide autocrine and paracrine stimuli, respectively, that activate PLC in vivo to advance the embryonic developmental program and coordinate it with that of the uterus .

Although PLC also generates DAG to activate PKC, cavitation of mouse morulae is not stimulated by supplementation with synthetic DAG or phorbol ester, and PKC inhibitors do not prevent ethanol-induced acceleration of cavitation (Stachecki and Armant 1996a). However, PKC inhibition does delay the basal rate of cavitation, indicating a role for PKC in homeostasis of the developing embryos. More critical in the acceleration of embryo cavitation is CaM (Stachecki and Armant 1996b). Inhibition of CaM by W-7 attenuates the acceleration of blastocoel formation after treatment with Ca²⁺ ionophore. Moreover, W-7 causes a dose-dependent delay in the basal cavitation rate, providing strong evidence for a requirement of this signaling pathway in normal development. CaM regulates a plethora of signaling and enzymatic activities through its interactions with other proteins (Lu and Means 1993), which in turn control cell proliferation, secretion and other critical functions for development. The role of CaMKII in advancing the cell cycle (Lorca et al. 1994) could regulate the timing of other developmental events that might be linked to the cell cycle. The generation of a Ca²⁺ transient clearly increases cell numbers at the same time as cavitation is stimulated (Stachecki and Armant 1996b). Protein secretion or trafficking events could be critical for advancing the developmental program and positioning the embryo for subsequent interactions with its microenvironment. The role of CaM in regulating these processes has long been appreciated (Chamberlain et al. 1995; Chen et al. 1999). The intricacies of Ca²⁺ signaling and its downstream networks (Ashby and Tepikin 2002) could be particularly effective for coordinating de novo gene expression with intracellular protein trafficking and secretory activities that regulate the developmental program. Additionally, CaM-independent activation of proteins by Ca²⁺ could also play a role in advancing preimplantation development . Clearly, there remains much to be learned about the regulation of blastocyst formation by Ca²⁺ downstream signaling.

As the blastocoel emerges and expands, the blastomeres complete differentiation into several new lineages. The mature blastocyst includes the pluripotent stem cells of the epiblast, a superficial portion of the inner cell mass that becomes the extraembryonic primitive endoderm, and trophoblast cells that form the first transporting epithelium, the trophectoderm (Wiley 1988), in the outermost layer of the embryo (Cockburn and Rossant 2010). With the increasing complexity of the embryo and the impending implantation of the blastocyst, cell-cell communication is of primary importance. A signaling system controlled by genes involved in cell lineage specification and the regulation of pluripotency directs mouse blastocyst development (Cockburn and Rossant 2010). While there have so far been no revelations regarding intracellular Ca²⁺ in the signaling pathways controlling differentiation of the various cell types that comprise the blastocyst, there is evidence that supports a role for Ca²⁺ signaling in interactions of mouse trophoblast cells with the maternal reproductive tract (Armant et al. 2000). With more sophisticated approaches for monitoring intracellular Ca²⁺ in real time, a role in cell lineage allocation of the preimplantation embryo could eventually come to light.

The molecular dialogue between trophoblast cells and the maternal environment (bottom of Fig. 6.1) includes paracrine signaling by factors secreted into the reproductive tract and juxtacrine signaling between endometrial tissues and embryonic cells that come into contact as implantation commences (Armant 2005). As a result of this communication, the uterine tissue becomes receptive to embryo implantation and the trophectoderm differentiates from a polarized epithelium into extravillous trophoblast cells capable of tissue invasion. Elevation of the intracellular Ca²⁺ concentration in mouse blastocysts with 0.1 % ethanol exposure rapidly alters gene expression, including the pluripotency regulator MYC and others responsible for rapid cell proliferation (Rout et al. 1997; Leach et al. 1999). This finding is consistent with observations that cell division rates increase in embryos after a Ca²⁺ transient (Stachecki and Armant 1996b; Leach et al. 1993). Expression of MYC is required for preimplantation development (Paria et al. 1992) and could be particularly important downstream of Ca²⁺ in the regulation of embryonic stem cells and trophoblast stem cells within the blastocyst. Intracellular Ca²⁺ signaling within the trophoblast cells is a key requirement in their phenotypic transformation and in coordinating the timing of this process with the ongoing development of the endometrium.

Secretions of the zona-enclosed blastocyst, as well as those of the endometrium, stimulate preimplantation embryonic development during transit from the oviduct into the uterus (Armant et al. 2000). The secreted products include agonists of both receptor tyrosine kinases and GPCRs, which activate a wide variety of downstream signaling pathways and second messengers, including Ca²⁺. Several experimental paradigms are available for examining the effects of embryotrophic factors on trophoblast differentiation using mouse blastocysts cultured in vitro (Armant 2006). These approaches have been used to demonstrate a critical role for Ca²⁺ signaling at the blastocyst stage, beginning with the observation that pharmacological induction of a Ca²⁺ transient not only accelerates development in vitro, but results in significantly improved implantation rates when treated blastocysts are transferred to pseudopregnant dams (Stachecki et al. 1994a).

Trophoblast differentiation is regulated by cross talk between GPCRs and receptor tyrosine kinases. Exposure of morulae or blastocysts to LPA significantly accelerates extravillous differentiation and this effect is attenuated by BAPTA-AM (Liu and Armant 2004). However, inhibition of ERBB receptor tyrosine kinases will prevent LPA stimulation of trophoblast development, suggesting that receptor cross talk is required in addition to the IP3-mediated release of Ca²⁺ from intracellular stores. The LPA receptor and other GPCRs are known to activate ERBB family receptors by a variety of mechanisms, including transactivation of ERBB1 and ERBB4 by the heparin-binding EGF-like growth factor (HBEGF; Fig. 6.1) (Umata et al. 2001). Indeed, antagonizing HBEGF inhibits LPA-mediated stimulation of trophoblast differentiation (Liu and Armant 2004). Moreover, intracellular HBEGF, monitored by immunofluorescence microscopy, is observed trafficking to the apical surface of the trophectoderm within 45 min after exposure to LPA. It is released from the surface within another 15 min. HBEGF is a type 1 transmembrane protein that requires cleavage by metalloproteinases for secretion of its extracellular domain, which is then free to bind ERBB1/4 during autocrine signaling (Holbro and Hynes 2004).

Calcitonin is a hormone of the parafollicular cells of the thyroid gland that is also produced in the uterus just prior to implantation in response to surging progesterone (Ding et al. 1994). Antisense inhibition of calcitonin mRNA expression in the uterus disrupts receptivity for blastocyst implantation (Zhu et al. 1998a, b). Like LPA, calcitonin receptors are coupled to G proteins that mobilize cytoplasmic free Ca²⁺ (Martin et al. 1995). In rodents, uterine expression of calcitonin begins on gestation day 3, increasing by day 4 (Ding et al. 1994), while its receptor is highly expressed by preimplantation embryos (Wang et al. 1998). Embryos first respond to calcitonin at the 4-cell stage by producing an intracellular Ca²⁺ transient. Blastocysts treated for only 30 min with calcitonin go on to form trophoblast outgrowths, a sign of trophoblast differentiation, significantly earlier than control embryos (Fig. 6.1). Additionally, calcitonin-treated blastocysts exhibit a precocious onset of fibronectin binding activity on the surface of the trophectoderm, and trafficking of the integrin subunit α5 (ITGA5) to the apical surface, both indicators of adhesion competence. Pretreatment of embryos with the intracellular Ca²⁺ chelator BAPTA-AM prevents both production of a Ca²⁺ transient and acceleration of trophoblast differentiation after calcitonin exposure (Wang et al. 1998).

The mechanism of HBEGF-induced stimulation of trophoblast development (Fig. 6.1) suggests a hypothesis to explain the requirement for its transactivation downstream of LPA. HBEGF accelerates trophoblast differentiation and induces ITGA5 trafficking in the absence of a GPCR agonist (Wang et al. 2000). Activation of the trophoblast is associated with, and dependent on, the production of a prolonged Ca²⁺ transient. Since the gamma isoforms of PLC (PLCγ1 and PLCγ2) can be activated by tyrosine phosphorylation (Meisenhelder et al. 1989; Margolis et al. 1989), it is possible that IP3-mediated release of Ca²⁺ from the ER produces the observed Ca²⁺ transient and acceleration of trophoblast differentiation; however, neither process is affected by HBEGF in the presence of a PLC inhibitor. Conversely, chelation of extracellular Ca²⁺ effectively blocks both the elevation of intracellular Ca²⁺ and the accelerated development (Wang et al. 2000). Furthermore, treatments with a panel of Ca²⁺ channel blockers demonstrated a requirement for N-type voltage-gated Ca²⁺ channels. It appears that HBEGF induces Ca²⁺ influx to sustain Ca²⁺ signaling and provide Ca²⁺ for replenishment of the ER at the end of the transient. The Ca²⁺ influx mechanism could therefore be essential after release of Ca²⁺ from the ER by GPCRs and might explain the cross talk between LPA or other GPCR agonists and the EGF signaling system. Voltage-gated Ca²⁺ channels have an important role during blastocyst development, demonstrated by implantation failure of blastocysts treated with high concentrations of endocannabinoids that inhibit voltage-gated channels (Wang et al. 2003).