Intracellular Ca²⁺ Signaling and Preimplantation Development

Abstract

The key, versatile role of intracellular Ca²⁺ signaling during egg activation after fertilization has been appreciated for several decades. More recently, evidence has accumulated supporting the concept that cytoplasmic Ca²⁺ is also a major signaling nexus during subsequent development of the fertilized ovum. This chapter will review the molecular reactions that regulate intracellular Ca²⁺ levels and cell function, the role of Ca²⁺ signaling during egg activation and specific examples of repetitive Ca²⁺ signaling found throughout pre- and peri-implantation development. Many of the upstream and downstream pathways utilized during egg activation are also critical for specific processes that take place during embryonic development. Much remains to be done to elucidate the full complexity of Ca²⁺ signaling mechanisms in preimplantation embryos to the level of detail accomplished for egg activation. However, an emerging concept is that because this second messenger can be modulated downstream of numerous receptors and is able to bind and activate multiple cytoplasmic signaling proteins, it can help the coordination of development through up- and downstream pathways that change with each embryonic stage.

6.1. Cellular Regulation of Cytoplasmic Ca²⁺ Concentrations

Ca²⁺ is a powerful second messenger required for cellular homeostasis that also regulates developmental processes (Berridge et al. 2003). Transient shifts in the concentration of cytoplasmic free Ca²⁺ direct several key events at the commencement of mammalian embryonic development. The partitioning and redistribution of Ca²⁺ among intracellular compartments contributes to a variety of mechanisms during fertilization and embryogenesis that control gene expression, mitochondrial activity, motility, secretion and protein trafficking, as detailed in a recent review (Miao and Williams 2012).

Indicative of Ca²⁺ signaling within cells are changes in the concentration of cytoplasmic free Ca²⁺ (Tsien and Tsien 1990), which can be monitored in real time using a variety of Ca²⁺-sensitive intracellular probes (Tsien 1988). Intracellular Ca²⁺ stored within the endoplasmic reticulum (ER) is released into the cytosol to generate downstream signaling (Berridge et al. 2003). Additionally, extracellular Ca²⁺ can enter the cytoplasm by crossing the plasma membrane. In either case, a multitude of Ca²⁺ channels mediate Ca²⁺ entry into the cytosol, while it is transported back into the ER or out of the cell by Ca²⁺ exchangers and pumps, resulting in a transient increase in cytoplasmic free Ca²⁺. Ca²⁺ signals are ultimately generated both by the release of stored Ca²⁺ from the ER and the influx of extracellular Ca²⁺ across the plasma membrane.

Ca²⁺ is released from the ER through two families of Ca²⁺ channels; inositol-1,4,5-trisphosphate (IP3) receptors (ITPR1–3) and the ryanodine receptors (RYR1–3) (Berridge et al. 2003). The sarco(endo)plasmic reticulm Ca²⁺-ATPases (SERCA1–3) provide a pump to replenish Ca²⁺ in the ER. While the RYRs are activated by cytoplasmic Ca²⁺, itself, the ITPRs are activated by IP3, which is generated enzymatically along with diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (PIP2) by any one of 13 phosphoinositide-specific phospholipase C (PLC) isoforms. PLC activity can, in turn, be regulated through a wide array of upstream pathways that involve G-protein-coupled receptors (GPCR), small GTPases and tyrosine kinases, as well as free Ca²⁺.

Ca²⁺ enters the cell through various plasma membrane channels to elevate cytosolic Ca²⁺ in response to upstream signals, as well as to sustain the capacity for Ca²⁺ signaling since plasma membrane Ca²⁺-ATPases (PMCAs) and the Na⁺/Ca²⁺ exchanger export it from the cell (Berridge et al. 2003). In excitable cells, voltage operated channels (VOCs) generate rapid Ca²⁺ influxes in response to membrane depolarization, and involve about 10 different channel proteins that comprise five VOC types (L, P/Q, N, R, T). Elevated cytoplasmic Ca²⁺ can, in turn, excite RYRs to trigger release from the ER as a mechanism of signal amplification. Additionally, there are Ca²⁺ channels operated by receptors (e.g., N-methyl-D-aspartate receptor, ATP receptor), second messengers (e.g., cAMP, arachidonic acid) and stretch, providing sensitivity to a diverse array of stimuli. Finally, the entry of extracellular Ca²⁺ is generally induced by the depletion of intracellular stores in a process referred to as capacitative or store-operated Ca²⁺ entry (SOCE) that ensures optimal refilling of the ER, and can extend cytosolic Ca²⁺ transients (Parekh and Putney 2005). SOCE is activated by STIM1, a sensor of ER Ca²⁺ concentration that physically communicates with the plasma membrane protein ORAI1, which then forms a Ca²⁺-selective pore (Muik et al. 2012).

There are a vast number of Ca²⁺-interacting proteins that become activated or inhibited as the Ca²⁺ concentration changes in the cytosol or in particular organelles. The generation of Ca²⁺ transients will therefore impact structural proteins, metabolism, signaling pathways and gene expression, which all play important roles in development. The sections which follow review some of the cellular events that Ca²⁺ signaling regulates during mammalian fertilization and preimplantation embryogenesis. It will become clear that Ca²⁺ signaling mechanisms arise not only during fertilization, but throughout development leading to blastocyst implantation, and that they regulate some of the key cellular events of the preimplantation phase (Fig. 6.1).

Fig. 6.1.

Calcium signaling during fertilization and preimplantation development. Gametes and embryos are depicted at various stages of mammalian development to illustrate proposed roles for Ca²⁺ signaling. The process begins with gamete fusion and delivery of PLCζ by the sperm to initiate a series of Ca²⁺ transients responsible for the cortical reaction (exocytosis of the red vesicles shown), followed by extrusion of the second polar body and formation of two pronuclei. The approximate number of Ca²⁺ transients required for post-fertilization events are indicated to the right. Autocrine signaling and interactions with the reproductive tract during embryonic development extend the role of Ca²⁺ signaling. For example, PAF generates Ca²⁺ transients during the early cleavage stages that lead to CaMKII activation, which prevents apoptosis through downstream CREB activation. Speculation (?) suggests that the secretion of growth factors from the embryo and EGA could also be dependent on this signaling by Ca²⁺. Ca²⁺ signaling continues to regulate events leading to implantation. Examples are shown of proposed roles in cavitation and trafficking of integrins to the surface in response to LPA, CT, HBEGF and FN-induced integrin signaling as the blastocyst acquires competence for implantation. Details of these events are discussed in the article. Abbreviations: CaMKII, Ca²⁺/calmodulin kinase II; CT, calcitonin; CREB, cAMP responsive element binding protein; EGA, embryonic genome activation; FN, fibronectin; HBEGF, heparin-binding EGF-like growth factor; ITGA, integrin α subunit; LPA, lysophosphatidic acid; MAPK, mitogen activated protein kinase; PAF, platelet activating factor; PLC, phospholipase C

6.2. Intracellular Ca²⁺ Signaling During Fertilization

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 decon-densing 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.

6.3. Regulation of Cleavage Stage Development by Intracellular Ca²⁺

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.

6.4. Regulation of Blastocyst Formation by Intracellular Ca²⁺

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 cavi-tate 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.

6.5. Regulation of Trophoblast Adhesion by Intracellular Ca²⁺

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).

As development continues, the embryo attaches to the uterine epithelium, resulting in apoptosis of the epithelial cells in contact with the blastocyst, and trophoblast invasion into the decidua (Carson et al. 2000). Cell-cell contact between the trophectoderm and uterine epithelium signals those cells to prepare for implantation. In addition to autocrine signaling by HBEGF in the blastocyst, HBEGF secreted by the embryo can initiate uterine decidualization (Paria et al. 2001; Hamatani et al. 2004), and induce localized uterine HBEGF expression in the region immediately surrounding the blastocyst (Das et al. 1994). Transmembrane HBEGF on the surface of uterine epithelial cells is capable of binding to ERBB1/4 receptors on the blastocyst, both to mediate juxtacrine signaling and contribute to the attachment of the blastocyst (Raab et al. 1996). However, the ability of HBEGF to activate ERBB4 signaling in the trophoblast requires homophilic binding of the adhesion molecule, trophinin, expressed on both the uterine epithelium and trophectoderm (Armant 2011). Binding between the opposing trophinin proteins dissociates a complex formed in trophoblast cells between the cytoplasmic domain of trophinin, its cytoplasmic partner, bystin, and transmembrane ERBB4 that inhibits its tyrosine kinase activity (Sugihara et al. 2007). The dissociated ERBB4 is then available for HBEGF-induced downstream signaling. Trophinin-trophinin binding simultaneously dissociates a complex between trophinin and PKCδ in the uterine epithelial cells, allowing PKCδ to become phosphorylated and enter the nucleus where it initiates apoptosis (Tamura et al. 2011). Thus, the cellular barrier to trophoblast invasion is removed and HBEGF is able to activate ERBB4, induce Ca²⁺ signaling and initiate trophoblast invasive differentiation precisely when the embryo first contacts the uterine wall.

Signaling pathways downstream of intracellular Ca²⁺ that regulate trophoblast differentiation have been examined in blastocysts exposed to calcitonin and HBEGF (Wang et al. 1998, 2000). Acceleration of trophoblast differentiation by calcitonin is not perturbed by protein kinase A inhibition, suggesting that cyclic-AMP has no role in this activity. However, HBEGF is unable to advance blastocyst differentiation in embryos treated with inhibitors of PKC or CaM. There appears throughout early development a pattern of events regulated by Ca²⁺ and CaM capable of inducing both new gene expression and waves of protein trafficking or secretion.

6.6. Regulation of Trophoblast Invasion by Intracellular Ca²⁺

After the attachment reaction between the mouse blastocyst and uterine epithelium, differentiated trophoblast cells migrate through apposing epithelial cells that undergo localized cell death (Welsh and Enders 1991; Parr et al. 1987) and adhere to the basement membrane in preparation for invasion of the underlying stroma on embryonic day 5 (Blankenship and Given 1992). At this point, the adherent trophoblast cells dissociate from the trophectoderm and commence invasion. Information is likely exchanged locally to coordinate the transformation of trophoblast cells into migratory, invasive cells at the time of blastocyst contact with the endometrial basement membrane. Cell adhesion molecules contribute to this coordination owing to their interaction with cytoplasmic proteins that produce second messengers, including Ca²⁺.

Integrins are transmembrane proteins that regulate cell adhesion and migration (Giancotti and Ruoslahti 1999; Hynes 2002). There are at least 28 integrin heterodimeric complexes composed of 18 alpha and 8 beta subunits. They are capable of initiating intracellular signaling upon binding to components of the extracellular matrix (ECM) or other ligands. ECM proteins, such as fibronectin, laminin and collagen, initiate integrin signaling at focal adhesion complexes composed of aggregated integrins and cytoplasmic signaling proteins. PLCγ, an isoform activated by tyrosine phosphorylation, can be included in this signaling complex to produce Ca²⁺ signaling (Jones et al. 2005; Tvorogov et al. 2005).

During human placentation, integrin expression switches between non-motile cytotrophoblast cells of the chorionic villi and the highly invasive extravillous tropho-blasts in the anchoring villi and decidua basalis (Damsky et al. 1994; Zhou et al. 1997). However, during the relatively brief time when the blastocyst transits from free-floating to implanting, a more rapid response to the changing microenvironment could be necessary. There is evidence that integrin signaling provides a sensory apparatus that instructs the trophectoderm as to the availability of ECM components through a post-transciptional mechanism that regulates the trafficking of integrins with appropriate functionality into the plasma membrane of trophoblast cells (Wang and Armant 2002; Armant 2005). Blastocyst exposure to the ECM proteins, fibronectin or laminin, stimulates adhesion within 1 h, but only to either fibronectin or laminin, respectively (Wang et al. 2002), demonstrating the ability of trophoblast ECM sensors to specifically recruit adhesive mechanisms appropriate to the local ECM composition.

Intracellular Ca²⁺ integrates cytoplasmic integrin signaling with the changing adhesive properties of trophoblast cells in the peri-implantation blastocyst (Wang et al. 2002). Transduction of the intracellular signal initiated by trophoblast contact with fibronectin is mediated through the integrins ITGA5/ITGB1 and ITGAV/ITGB3. Downstream of integrins, the intracellular Ca²⁺ concentration rapidly increases as a transient or set of oscillations that are sustained for up to 1 h (Fig. 6.1). Accordingly, BAPTA-AM prevents the ligand-mediated enhancement of adhesion to fibronectin. Pharmacologic elevation of intracellular Ca²⁺ by ionomycin stimulates adhesion to fibronectin in the absence of any exposure to the ligand, suggesting that Ca²⁺ signaling is sufficient and necessary for integrin signaling and fibronectin sensing by trophoblast cells.

During integrin signaling in trophoblast cells, Ca²⁺ is released from intracellular stores regulated by IP3. Unlike the pathway downstream of HBEGF (Wang et al. 2000), inhibition of voltage gated Ca²⁺ channels or non-voltage gated Ca²⁺ channels to prevent Ca²⁺ influx fails to prevent the production of a Ca²⁺ transient or the increased adhesiveness of trophoblast cells after exposure to fibronectin (Wang et al. 2007). However, the PLC inhibitor U73122 or the ITPR antagonist Xestospongin C are each capable of preventing intracellular Ca²⁺ release and the change in adhesiveness of trophoblast cells upon fibronectin treatment. This mechanism is more similar to Ca²⁺ mobilization by GPCR agonists. The PLCβ family is activated by G-proteins (Rebecchi and Pentyala 2000), but the G-protein inhibitor suramin does not interfere with the stimulation of trophoblast adhesion by exposure to fibronectin (Wang et al. 2007). However, the tyrosine kinase inhibitor genistein interferes with both Ca²⁺ release and increased adhesiveness, suggesting that a tyrosine kinase-dependent PLC isoform could regulate Ca²⁺ release. Genistein did not prevent ligand-mediated upregulation of adhesion to fibronectin when embryos were stimulated with ionomycin (J. Wang and Armant, unpublished), indicating that tyrosine kinase activity is specifically required upstream of Ca²⁺ mobilization. The phosphotyrosine-dependent isoforms, PLCγ1 and PLCγ2 (Rebecchi and Pentyala 2000), associate with focal adhesion complexes and become activated by focal adhesion kinase, src or other protein tyrosine kinases localized in focal adhesions (Jones et al. 2005; Tvorogov et al. 2005; Watson et al. 2005). Mouse blastocysts express both isoforms of PLCγ and upon stimulation of the blastocyst with fibronectin there is rapid phosphorylation of PLCγ2 at the apical surface of trophoblast cells (Wang et al. 2007). These findings suggest that fibronectin sensing by the trophoblast involves the formation of focal adhesions containing PLCγ2 and protein tyrosine kinases that activate it to induce Ca²⁺ signaling. Whether exposure of the trophoblast to different ECM proteins, such as laminin, induces the same response is not known, but some divergence upstream or downstream of Ca²⁺ would be anticipated to upregulate laminin binding rather than fibronectin binding activity, as is demonstrated to occur (Wang et al. 2002).

Proteins that mediate integrin signaling downstream of Ca²⁺ include CaM and PKC (Wang et al. 2002), resulting in the insertion of new integrins, including ITGA2B/ITGB3 (Fig. 6.1), in the plasma membrane (Rout et al. 2004). While signaling upstream of Ca²⁺ can be considered as “outside-in” signaling, “inside-out” signals result in changes to integrin function, with insertion of new integrins or activation of those already in the plasma membrane (Hynes 1992). Adhesion of trophoblast cells is not increased when blastocysts exposed to fibronectin are treated with inhibitors of either CaM or PKC (Wang et al. 2002). Protein trafficking and secretion are regulated by CaM and PKC in cells downstream of Ca²⁺ signaling (Chen et al. 1999; Peters and Mayer 1998; Quetglas et al. 2000), and ligand-medated upregulation of trophoblast adhesion to fibronectin is sensitive to inhibition of protein trafficking by brefeldin A (Schultz and Armant 1995). Indeed, trafficking of the integrin ITGA2B to the trophectoderm surface is associated with changes in trophoblast adhesion and migration (Rout et al. 2004).

6.7. Conclusions and Speculation

The regulation of numerous cellular processes during preimplantation development by intracellular Ca²⁺ (Fig. 6.1) indicates the potential magnitude of its importance as a recurring mechanism. During egg activation, Ca²⁺ operates predominantly through CaM and PKC to induce cortical granule exocytosis, advance the cell cycle and initiate new transcription. Similar activities are associated with Ca²⁺ at subsequent stages of pre- and peri-implantation development, although this period has not been as thoroughly studied as fertilization. During the early cleavage stages, PAF advances the cell cycle through Ca²⁺ and CaM. Cavitation is stimulated by additional autocrine and paracrine signaling through a similar mechanism, and there is evidence that compaction depends on cytoskeletal changes regulated by CaM and other Ca²⁺ binding proteins of the cytoskeleton. The same signaling mechanism operates during implantation downstream of growth factors and ECM components that advance blastocyst development and trophoblast invasion. Although the molecular linkage between Ca²⁺, CaM and early developmental events remains to be clarified, it has been established that the second messenger Ca²⁺ can activate new sets of genes, promote cell proliferation and alter the composition of the plasma membrane by transducing signals originating from the external environment.

The responsiveness of the preimplantation embryo to its surroundings is well established and essential to coordinate its development with that of the genetically distinct maternal tissues. The embryo passes through the stages of cavitation, attachment to the endometrial epithelium and invasion of the decidua much faster in vivo than during embryo culture ex vivo, suggesting that the maternal environment provides a milieu that accelerates trophoblast development (Armant 2005). Preimplantation development in the absence of maternal support could perturb the embryonic epigenome, which might ultimately impede both fetal and placental development (Chason et al. 2011). DNA methylation, demethylation and histone modification during early embryogenesis are highly sensitive to the maternal environment, influencing subsequent gene expression throughout embryonic and adult life. The epigenome could, therefore, be essential for the correct expression of genes that mediate autocrine signaling in the developing preimplantation embryo, as well as genes that respond to cues from the reproductive tract. It can be hypothesized that Ca²⁺ signaling links extrinsic cues—a sperm, a growth factor, a uterine cell, the ECM—to downstream pathways that initiate cell division, new gene expression, cell shape change, intracellular trafficking or cell migration, which advances embryonic development and prepares cells to respond to subsequent events. This concept is exemplified during egg activation by a series of diverse cellular events driven by sequential oscillations of Ca²⁺. Extrapolation to the preimplantation period would suggest that elevation of Ca²⁺ by extrinsic (or autocrine) factors could activate transcription factors that induce new gene expression to advance development. At the same time, Ca²⁺ signaling could stimulate trafficking of new receptors to the plasma membrane and trigger secretion of embryotrophic factors. With repeated cycles of signaling, not only does the developing embryo change, but also the molecular signaling mechanisms at its disposal. As the blastocyst contacts the uterine wall, additional rounds of Ca²⁺ transients in response to cell-cell or cell-ECM contact can induce trophoblast differentiation to an adhesion competent state, setting the stage for invasion of the decidua. It has been hypothesized (Armant 2005) that Ca²⁺ signaling is a homeostatic requirement for successful preimplantation development and blastocyst implantation due to its repetitive role integrating extrinsic information into the intrinsic developmental program. Support for this idea awaits a more thorough understanding of the signaling networks that operate in preimplantation embryos downstream of cytoplasmic Ca²⁺.