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Phosphatidylinositol 3-kinase signaling in mammalian preimplantation embryo development

Disciplines of Medicine and Physiology, Human Reproduction Unit, Royal North Shore Hospital, University of Sydney, St Leonards, New South Wales 2065, Australia

Correspondence should be addressed to C O'Neill; Email: chriso{at}med.usyd.edu.au

	Abstract

Top Abstract Introduction Synthesis of 3'... Evidence for a role... Action of embryotrophic ligands... Activation of PI3 kinase... Downstream effectors of PIP3... Conclusion Declaration of interest References

 
The development of the preimplantation mammalian embryo is an autopoietic process; once initiated development proceeds without an absolute requirement for external information or growth cues. This developmental autonomy is partly explained by the generation of autocrine trophic ligands that are released and act back on the embryo via specific receptors. Several embryotrophic ligands cause receptor-dependent activation of 1-o-phosphatidylinositol 3-kinase. This enzyme phosphorylates phosphatidylinositol-4,5-bisphosphate to form phosphatidylinositol-3,4,5-trisphosphate. Genetic or pharmacological ablation of this enzyme activity disrupts normal development of preimplantation embryos. Phosphatidylinositol-3,4,5-trisphosphate is a membrane lipid that acts as a docking site for a wide range of proteins possessing the pleckstrin homology (PH) domain. Such proteins are important regulators of cell survival, proliferation, and differentiation. RAC- serine/threonine protein kinase is an important PH domain protein and its activity is required for normal preimplantation embryo development and survival. The activity of a range of PH domain proteins is also implicated in the normal development of the embryo. This review critically examines the evidence for the activation of 1-o-phosphatidylinositol 3-kinase in the generation of pleiotypic trophic response to embryotrophins in the autopoietic development of the preimplantation embryo.

	Introduction

Top Abstract Introduction Synthesis of 3'... Evidence for a role... Action of embryotrophic ligands... Activation of PI3 kinase... Downstream effectors of PIP3... Conclusion Declaration of interest References

 
The generation of intracellular signals in response to extracellular stimuli is an essential function of cells. In metazoa, cellular responses to extracellular cues are central to maintaining ordered and coordinated cellular behavior within the complex spatiotemporal environment of the discrete organism. As such, appropriately controlled responses to external cues are critical to the normal development of the embryo. Some ancient forms of secondary messaging (e.g., calcium and cyclic AMP) seem to be universal. Other forms of signaling have been elaborated primarily in association with the development of multicellular eukaryotes. An example of this is the generation by 1-o-phosphatidylinositol 3-kinase (PI3 kinase) of phosphatidylinositol (PtdIns) that is phosphorylated at the 3'-carbon of the inositol ring (Michell 2007).

The preimplantation mammalian embryo can develop in vitro in the absence of all exogenous vitamins, growth factors, or hormones. This apparent developmental autonomy distinguishes the early embryo from all cells at the later stages of development and postnatally, where normal cells have a requirement for exogenous ligands. This autonomy seems to be explained by the action of trophic ligands that are both released by and act on the embryo. These ligands are classed as autocrine since the responding cells also produce (and release) the factor (O'Neill 2008).

A curious feature of the action of these ligands is that their relative deprivation does not create an obvious block of mitosis; rather there is a generalized loss of viability, resulting in the progressive death of cells comprising the embryo. On the basis of this response, it seems that an important role of autocrine ligands was to promote the survival of embryonic cells (reviewed in O'Neill 2008).

The 3'-phosphorylated PtdIns generated by PI3 kinase is known to play critical roles in cell survival, and recent evidence indicates that they play an important role mediating the actions of trophic ligands on the preimplantation mammalian embryo. This paper provides a general overview of the biochemistry and roles of the 3'-phosphorylated PtdIns and critically reviews the present progress in assessing their roles in early mammalian embryo development.

	Synthesis of 3'-phosphoinositides

Top Abstract Introduction Synthesis of 3'... Evidence for a role... Action of embryotrophic ligands... Activation of PI3 kinase... Downstream effectors of PIP3... Conclusion Declaration of interest References

 
Inositol is present in membrane lipids in a number of forms. In the context of the present discussion, the most important form is myo-inositol, present as 1,2-diacyl-sn-glyceryl-3-phosphatidylinositol (PtdIns; structure is shown in Box 1). Interest in the possible signaling roles of this class of lipid was ignited with the discovery that the potent mitogen, platelet-derived growth factor, caused a tyrosine kinase-dependent production of a novel polyphosphoinositide. This was identified as a 1,2-diacyl-sn-glyceryl-3-PtdIns-3-phosphate (Auger et al. 1989). This discovery followed on the observations that the oncogenic properties of polyoma virus required the actions of a phosphatidylinositol-3 kinase enzyme activity (Whitman et al. 1988). It was subsequently found that this enzymatic activity (age-1) in C. elegans was important in regulating the life span of this organism (Morris et al. 1996).

The generation of PtdIns-3,4,5-P (PIP3) as a consequence of ligand–receptor interaction is generally considered to primarily involve the pathway as follows: PtdIns-4-P (PIP)PtdIns-4,5-P (PIP2)PtdIns-3,4,5-P (PIP3) (Box 1). Within this pathway, the action of PI3 kinase is generally the rate-limiting step. Consequently, it is the action of PI3 kinase which receives most attention when considering the activity of this signaling pathway.

A systematic investigation of the biosynthesis of PtdIns by the preimplantation embryo has not been undertaken. For a number of species, the addition of inositol to culture media improves embryo development (Kane 1988, 1989, Kane & Bavister 1988, Fahy & Kane 1994, Hynes et al. 2000, 2002), suggesting that de novo synthesis of myo-inositol may be limiting during this stage of development (at least in vitro). Addition of inositol to culture media resulted in a 9.9-fold increase in thymidine incorporation into DNA and a 3.6-fold increase in amino acid incorporation into protein for rabbit morulae cultured through to expanded blastocysts (Fahy & Kane 1992). In cattle embryos, [³H]myo-inositol was taken up by two-cell and four-cell embryos, morulae, and early blastocysts in a sodium-dependent manner, and was incorporated into PtdIns, PIP, and PIP2 (Hynes et al. 2000). PIP3 was not detected in this analysis, but this may be due to the very transient nature of its synthesis under normal circumstances.

	Evidence for a role of PI3 kinase in early embryo development

Top Abstract Introduction Synthesis of 3'... Evidence for a role... Action of embryotrophic ligands... Activation of PI3 kinase... Downstream effectors of PIP3... Conclusion Declaration of interest References

 
Three classes of PI3 kinases have been defined (classes I, II, and III; reviewed in Fruman et al. 1998). Members of the class I PI3 kinases are considered to be most relevant in the generation of PIP3 in response to external trophic ligands. Class II molecules are large (170–210 kDa) molecules that may preferentially phosphorylate PtdIns and PtdIns-4-P. Class III kinases phosphorylate PtdIns to produce PtdIns-3-P. The generation of this lipid has important roles in membrane trafficking and organelle structure and function (Gill et al. 2007). Class I PI3 kinases are the most important in the formation of PIP3, and only this class will be considered in this review.

Class I PI3 kinases (or 1-o-phosphatidylinositol-4,5-bisphosphate 3-kinase) are formed by heterodimerization of p85/p101 regulatory and p110 catalytic subunits. There are several forms of these subunits (p85 and β; p101; p110 , β, and ). Dimers containing p85 subunits are referred to as class Ia and those with p101 as class Ib (Fruman et al. 1998). The expression of multiple subunits of class Ia PI3 kinase genes were detected in the two-cell mouse embryo by reverse transcriptase PCR, and included p85 and β, and p110, β, , and (Lu et al. 2004). Immunofluorescence analysis showed expression of p85 and p110 subunits from the one-cell stage through to the blastocyst stage, although the subclass of each of the subunits has not been determined (Riley et al. 2005). The expression by the embryo of the p110 (class Ib) subunit has not been investigated. Despite the apparently promiscuous expression of many members of this enzyme class, only the p110β catalytic subunit is implicated as essential for normal preimplantation embryo development. Deletion of p110β (Pik3cb^–/–) catalytic subunit of the enzyme results in early embryonic lethality. After mating of mice heterozygous for the deleted allele (Pik3cb^+/–), most of the resulting homozygous null embryos died prior to the blastocyst stage. A small proportion persisted until implantation, but resorbed soon thereafter (Bi et al. 2002). Deletion of the other forms of the catalytic subunit did not result in a preimplantation phenotype. Homozygous deletion of the p110 (Pik3ca^–/–) caused death in utero between E9.5 to E10.5 (Bi et al. 1999). Mice expressing a catalytically inactive form of p110 (Pik3cd ^D910A/D910A) were viable but had impaired antigen receptor signaling in B and T cells and they also developed inflammatory bowel disease (Okkenhaug et al. 2002). Deletion of p110 (Pik3cg^–/–) resulted in normal viability, longevity, and fertility, but mice had defects in innate immune and inflammatory responses (Chang et al. 2007).

Mice deficient in any form of the regulatory subunit survive to implant and show apparently normal development. Those null for p85 (Pik3r1^–/–) die within days after birth and have impaired B-cell development (Fruman et al. 2000). Embryos null for p85β (Pik3r2^–/–) are viable but show hypoinsulinemia, hypoglycemia, and improved insulin sensitivity after birth (Ueki et al. 2002). The absence of a preimplantation phenotype for the regulatory subunits may infer that p110β can dimerize with any of the available regulatory subunits, and that redundancy at this level occurs during early embryo development. The results of these studies show an essential role for the p110β catalytic subunit and an indeterminant role for the regulatory subunits of PI3 kinase in preimplantation embryo development.

An alternative approach to defining the role of PI3 kinase-mediated signals is the use of reversible pharmacological inhibitors. These have the advantage (and limitation) of acting across the range of PI3 kinase classes and subclasses; thus issues of potential redundancy between different subunits are averted. Two structurally unrelated inhibitors of PI3 kinase are widely used for investigating its action; the fungal metabolite wortmannin and the synthetic inhibitor LY294002. Wortmannin acts as a selective, cell permeable, and irreversible inhibitor of PI3 kinase at concentrations below 50–100 nM and has an IC₅₀ of 5–10 nM for different cell types (Fruman et al. 1998). At the micromolar dose range, wortmannin also inhibits myosin light chain kinase, phosphatidylinositol 4-kinase, MAP kinase, and phospholipase D (Backer 2000). LY294002 is a cell permeable, potent, and selective PI3 kinase inhibitor that acts on the ATP-binding site of the enzyme (IC₅₀ 1.5–3 µM); it should be used at concentrations of less than 30 µM. It can, for example, non-selectively inhibit DNA-dependent protein kinase and mammalian target of rapamycin (mTOR) at high micromolar concentrations (Backer 2000). Thus, as with all pharmacological agents, selectivity of action can only be assumed over a narrow, defined concentration range. Some studies of PI3 kinase in embryos have used inhibitors outside their selective concentration range and conclusions based on those studies should be viewed with caution. In general, a conclusion that an effect is due to the actions of PI3 kinase should be made on the basis of it being blocked by both wortmannin and LY294002, when both are used within their selective dose range. However, PI3 kinase also possesses a serine kinase activity that is also inhibited by wortmannin and LY294002. Hence, definitive evidence for the action of PI3 kinase via its lipid kinase activity also requires more direct evidence of the generation and action of PIP3.

Both wortmannin and LY294002 reduced, in a dose-dependent manner (10–50 nM and 3–15 µM respectively), the rate of development of zygotes into normal blastocysts (Lu et al. 2004). This was accompanied by a reduction in the number of cells within embryos and an increase in the number of cells with signs of cell death. Acute treatment of mouse blastocysts with LY294002 (50 µM) induced an increased incidence of apoptosis (TUNEL staining) within 10 h of treatment (Gross et al. 2005). Using the same model, neither inhibition of MAP kinase (U0126) nor mTOR (rapamycin) induced a marked increase in TUNEL staining, suggesting that the effect of this concentration of LY294002 on cell survival may have been due to selective inhibition of PI3 kinase (Gross et al. 2005). Another study showed that very high concentrations of LY294002 (250 µM) reduced the incidence of mouse blastocyst hatching in vitro (Riley et al. 2005).

	Action of embryotrophic ligands through PI3 kinase

Top Abstract Introduction Synthesis of 3'... Evidence for a role... Action of embryotrophic ligands... Activation of PI3 kinase... Downstream effectors of PIP3... Conclusion Declaration of interest References

 
A present paradigm of cell biology is that all cells require the action of a range of survival factors to maintain the latency of constitutively active effectors of cell death (Raff et al. 1994). In the absence of such survival factors, it is proposed that the cells enter into a default process of apoptotic cell death. It is argued that this is a mechanism for providing positional information to cells during development (and during subsequent life), which facilitates tissue remodeling and helps guard against ectopic or metastatic growth of cells. Survival signaling induced by trophic ligands has been studied in many somatic cell types and is commonly transduced by the action of PIP3. The actions of autocrine survival factors during the preimplantation stage of growth suggest a potential role for ligand-induced generation of PIP3 in the survival of early embryonic cells.

One well-described autocrine embryotrophin for the preimplantation embryo is Paf (platelet-activating factor, 1-o-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine) (O'Neill 2005, 2008). This bioactive phospholipid acts via a membrane receptor to induce characteristic calcium transients in the zygote and two-cell embryo (Emerson et al. 2000). The Paf receptor is known to activate PI3 kinase in some cell types (Honda et al. 2002). Both wortmannin (10–50 nM) and LY294002 (3–15 µM) significantly blocked the Paf-induced calcium transients in two-cell mouse embryos. LY303511 (15 µM, an inactive analogue of LY294002) had no effect of the Paf-induced calcium transients (Emerson et al. 2000). Buffering these calcium transients reduced the normal developmental capacity of zygotes to the blastocyst stage (Emerson et al. 2000). Insulin and the IGFs are also important trophins for the preimplantation embryo. Insulin-induced glucose uptake in mouse blastocysts was reduced by high concentration of wortmannin (100 nM) or LY294002 (250 µM) (Riley et al. 2005). In the blastocyst, insulin acts on the IGF1 receptor (Harvey & Kaye 1992) and this receptor is well known in other settings for its activation of PI3 kinase (Kotani et al. 1994).

	Activation of PI3 kinase by ligand receptors

Top Abstract Introduction Synthesis of 3'... Evidence for a role... Action of embryotrophic ligands... Activation of PI3 kinase... Downstream effectors of PIP3... Conclusion Declaration of interest References

 
Regulatory subunits of PI3 kinase possess Src homology 2 (SH2) domains that recognize the phosphorylated tyrosine motifs that are commonly created upon activation of ligand receptors (Fig. 1). The regulatory subunits also possess proline-rich motifs allowing them to bind a range of cytoplasmic kinases (Pleiman et al. 1994). The interaction of p85 with ligand-activated receptors allows binding of their p110 catalytic partner. The regulatory subunits can act to enhance/stabilize the activity of the catalytic subunit, but under some circumstances it can also act to reduce enzyme activity (Ueki et al. 2002). The p110 catalytic subunits can also be activated directly by receptor-activated Ras (Fruman et al. 1998). The p110 subunit can be activated by G-protein-coupled receptors, and it is possible that β-subunits of the G-protein interact directly with p110 (Stoyanov et al. 1995). Heterodimers containing p85/p110β are synergistically activated by both G-protein β and phosphotyrosyl peptides, indicating the potential for crosstalk between different receptors activating these processes (Katada et al. 1999). Insulin and insulin-like growth factor-I (IGF1) receptors act more indirectly on PI3 kinase. The activation of these receptors results in tyrosine autophosphorylation and the SH2 domains of insulin receptor substrate (IRS) proteins bind to these phosphorylated sites on the receptor. The bound IRS protein then binds p85 and p110 (Ueki et al. 2002).

View larger version (28K): [in this window] [in a new window]   Figure 1 Activation of PI3 kinase. Ligand activation of receptors induces structural changes, such as autocatalytic tyrosine phosphorylation. The p85 regulatory subunits of PI3 kinase are activated by ligand-bound receptors, typically by binding to newly phosphorylated tyrosine. The p110 catalytic subunits are activated by association with the p85 subunit, and this creates the active enzyme. The PIP3 generated typically has a very short life span within the cell due to its degradation by phosphatases such as PTEN.

Although the lipid kinase activity of PI3 kinase receives most attention, it is important to note that the enzyme also has an intrinsic serine kinase activity (Fruman et al. 1998). This protein kinase activity is also blocked by the common PI3 kinase inhibitors. The use of these inhibitors, therefore, does not distinguish between a response involving the protein or lipid kinase activities of PI3 kinase. The protein kinase activity is autocatalytic, and this is likely to be a major function; however, it can also phosphorylate unrelated target proteins, for example, IRS (Uddin et al. 2000).

To date, direct evidence for lipid kinase activity of PI3 kinase has only been demonstrated for the action of Paf. The Paf-induced calcium transient consists of an influx of external calcium and the release of IP₃-sensitive stores of internal calcium. LY294002 (10 µM) completely abolished the calcium influx induced by Paf in two-cell embryos (as assessed by manganese quench analysis) (Lu et al. 2003). The calcium transient induced by Paf was accompanied by a marked hyperpolarization of membrane potential and this was caused by a net outward ion current (280 pA). This current is composed of a 4,4'-diisothiocyanatostilbene-2,2'-disulfonate-sensitive (anion channel blocker) and tetraethylammonium chloride-sensitive (K+ channel blocker) channels (Li et al. 2007b, 2007c). Direct infusion of PIP3 (1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylinositol-3,4,5-trisphosphate) into the cytoplasm of the two-cell embryo mimicked the action of Paf in inducing the activity of this current and membrane hyperpolarization. Furthermore, infusion of a PIP3 blocking antibody (monoclonal RC6F8) into the two-cell embryo blocked the expression of this hyperpolarizing current and also blocked the Paf-induced calcium transients (heat-treated antibody had no effect on these responses to Paf) (Li et al. 2007b). These results show that Paf acted to induce the phospholipid kinase activity of PI3 kinase in the two-cell embryo.

Paf signaling is observed, and is most important, during the zygote and two-cell stages of development. This infers that the actions of Paf, via its receptor, may be sufficient to maintain the required level of PI3 kinase-mediated signaling up to the two-cell stage, but that other factors are required for PI3 kinase action after that time. This is consistent with observations of the PI3 kinase-dependent actions of other trophic ligands, including insulin (Navarrete Santos et al. 2004, 2008, Riley et al. 2005), IGF1 (Navarrete Santos et al. 2008), and transforming growth factor- (Kawamura et al. 2005, 2007) in the later stage preimplantation embryos. One study showed that treatment of mouse blastocysts with a PI3 kinase inhibitor (LY294002) induced a high rate of apoptosis and a decrease in both 2-deoxyglucose uptake and hexokinase activity (Riley et al. 2006). Yet, the study used concentrations of 250–1000 µM LY29002, which are well outside the concentration where selectivity of action of the drug can be assumed. This treatment is likely to have caused inhibition of a wide range of other kinases and related enzymes. It is doubtful if the result informs us of a role for PI3 kinase in the blastocysts. Another study (Navarrete Santos et al. 2008) showed that a lower concentration of LY294002 (25 µM) blocked the phosphorylation of RAC- serine/threonine protein kinase (AKT) that was induced by insulin or IGF1 in the day 6 rabbit blastocyst. At this concentration, the actions of LY294002 are likely to be selective, showing that the actions of insulin and IGF1 at physiological concentrations act in the blastocyst via PI3 kinase. Thus, throughout the preimplantation stage of development, a range of trophic ligands may cooperate to maintain a level of PI3 kinase signaling required for normal embryo development and survival.

	Downstream effectors of PIP3 signaling

Top Abstract Introduction Synthesis of 3'... Evidence for a role... Action of embryotrophic ligands... Activation of PI3 kinase... Downstream effectors of PIP3... Conclusion Declaration of interest References

 
Class I PI3 kinase preferentially converts PIP2 to PIP3 that serves as a docking site for proteins that possess the pleckstrin homology (PH) domain. There is also an unrelated domain (the FYVE domain) that binds to PtdIns-3-P. Since PtdIns-3-P is not a primary product of class I PI3 kinases, the actions of FYVE domain proteins will not be considered further (for a review on this domain see Gillooly et al. 2001).

The PH domain is one of the most common within the cellular proteome, and many proteins with important signaling functions possess it. The PH domain is 120 amino acids long and consists of two perpendicular anti-parallel β-sheets, followed by a C-terminal amphipathic helix (Lemmon et al. 1996). There can be considerable amino acid sequence differences within the PH domain between different proteins. Proteins of many different functional classes possess PH domains, including serine/threonine kinases, tyrosine kinases, GTPases, adaptor proteins, cytoskeletal-associated proteins, and (not surprisingly) phosphoinositide-metabolizing enzymes (Rebecchi & Scarlata 1998). The interaction between PIP3 and PH domains serve to tether the protein to regions of the membrane, which contain PIP3. The PH domain in effector proteins is commonly surrounded by hydrophobic regions of the protein. Thus, the PH domain targets the protein to PIP3 and the hydrophobic sites significantly enhance the affinity of binding to the membrane. In this way, the generation of PIP3 in response to extracellular ligands results in the recruitment of a range of PH-containing proteins to the membrane.

Binding of proteins in this way facilitates signaling in at least three ways: (1) binding of some proteins to PIP3 can induce conformational changes in the bound protein allowing, for instance, autophosphorylation of the protein (Standaert et al. 2001); (2) allosteric changes in the bound protein can result in changes to their function, such as the relief of pseudosubstrate autoinhibition (Standaert et al. 2001); and (3) binding can bring the PH domain substrate proteins into close proximity with the PH domain containing kinases, facilitating phosphorylation (Rebecchi & Scarlata 1998).

Two important signaling proteins that provide a well-studied example of this final type of interaction are 3-phosphoinositide-dependent protein kinase 1 (PDK) and AKT. Both these proteins possess PH domains and their association with PIP3 allows PDK to phosphorylate AKT. This phosphorylation facilitates the kinase activity of AKT (also known as protein kinase B; Fig. 2).

View larger version (23K): [in this window] [in a new window]   Figure 2 Downstream consequences of PIP3 formation. In the embryo, the best-characterized responses are to Paf. The diagram summarizes the demonstrated (black lines) or inferred (grey lines) responses to Paf in the early mouse embryo. The formation of PIP3 within the cytoplasmic surfaces of membranes creates a binding site for proteins containing the PH domain. Some proteins, such as PDK, autoactivate upon binding to PIP3 and can in turn phosphorylate neighboring PH domain proteins bound to PIP3, including AKT and a range of other effectors. The effectors can then exert actions on many downstream targets. PDK can also phosphorylate proteins that do not contain PH domains. These also have a range of independent downstream targets. The net outcome of PIP3 synthesis within a cell is the generation of a complex network of effectors that can act in concert to regulate cell survival. In the early embryo, the net result of PIP3 mediated signaling for the survival of the cells.

One important downstream target of calcium signaling is the cAMP-responsive element-binding protein (CREB) family of transcription factors. These transcription factors are activated by phosphorylation, which can be induced by calcium–calmodulin-dependent kinases (Shaywitz & Greenberg 1999, Mayr & Montminy 2001). In the two-cell embryo, calcium transients induce the phosphorylation and nuclear localization of CREB (Jin & O'Neill 2007). Nuclear localization of CREB was observed to increase for the first time at the mid-two-cell stage and again at the eight-cell stage. Nuclear localization of phosphorylated CREB was also most evident at these stages of development. Phosphorylation of CREB was apparently independent of the actions of cAMP, but required the actions of calcium and calmodulin (Jin & O'Neill 2007). Two members of the CREB family of transcription factors (CREB and activating transcription factor 1 (ATF1)) are expressed in the preimplantation embryo and are essential for normal preimplantation embryo development (Bleckmann et al. 2002). CREB and ATF1 are closely related by structure and function and show overlapping functions due to their capacity to both homo- and heterodimerize to form functional transcription factors. The dimerized transcription factor binds to consensus CREB response element (CRE) within the regulatory regions of genes. Preimplantation embryos with the compound mutations Creb1^–/–Atf1^–/– succumb to extensive apoptosis (Bleckmann et al. 2002), indicating an essential requirement of these transcription factors for normal embryo survival.

The transcriptome generated by CRE-mediated transcription is very extensive (Zhang et al. 2005). Importantly, this transcriptome includes many proteins that are necessary for survival signaling, including the anti-apoptotic mediator BCL2 (Fig. 2). The activation of CREB is well recognized to be an important component of the cells survival signaling mechanism (Walton & Dragunow 2000) and is required for survival of the preimplantation embryo. Activation of this transcription factor by calcium transients makes it a likely component of the embryo's pleiotypic response to ligand-mediated PIP3 production.

Another well-studied PIP3-sensitive protein that contains PH domains is AKT. The two-cell mouse embryo expresses the three genes that code for AKT (Akt1–3) (Li et al. 2007b) and immunostaining showed that AKT protein is expressed throughout the preimplantation stage (Riley et al. 2005, Li et al. 2007b). PIP3 can activate PDK, which in turn phosphorylates PIP3-bound AKT. Serine 473 phospho-AKT is detected in all stages of preimplantation development in the mouse (Riley et al. 2005), and exposure of two-cell embryos to Paf in vitro caused an upregulation in its expression (Li et al. 2007b). Acute treatment with insulin increased the level of phosphorylated AKT, as assessed by Western blot analysis in the mouse (Riley et al. 2005) or rabbit (Navarrete Santos et al. 2008). It is known that insulin and IGF1 improve mouse embryo survival in vitro, and that acting through the IGF1 receptor insulin promotes glucose transport-8 expression in the mouse blastocyst (Pinto et al. 2002). In the rabbit blastocyst, insulin induced phosphorylation of AKT in the trophectoderm but not in the inner cell mass, while IGF1 created the opposite response. This result indicates that lineage specificity of PI3 kinase signaling may occur in the early embryo, and highlights the context-specific nature of cellular responses to a given ligand.

Injection of mRNA coding for a constitutively active myristoylated AKT into mouse zygotes enhanced their rate of cell cycle progression; conversely mRNA of kinase-deficient AKT (Akt1-KD) delayed the entry of zygotes into mitosis. It was further shown that AKT induced the phosphorylation of cell division cycle 25 homolog B (CDC25B), and hence induced MPF activity (Feng et al. 2007). Two structurally unrelated inhibitors of AKT activity (deguelin, 5–15 nM; AKT inhibitor, 3–10 µM) induced a dose-dependent reduction in the rates of zygote development to the blastocyst stage in vitro (Li et al. 2007b). The embryopathy induced by AKT inhibition was partially reversed by the addition of exogenous Paf to culture medium. The similar inhibitory profiles of putative PI3 kinase (Lu et al. 2004) and AKT inhibitors (Li et al. 2007b) are consistent with these agents acting within the same signaling pathway in the early embryo. Furthermore, Paf partially reversed the adverse effects of both PI3 kinase and AKT inhibition on embryo development. AKT inhibitors did not affect the production of Paf-induced calcium transients (Li et al. 2007b). This indicates that activation of the calcium transient was independent of AKT activation.

Activated AKT can phosphorylate a wide range of downstream targets, many of which are required for normal cell survival (Datta et al. 1999, Lawlor & Alessi 2001, Downward 2004). Examples of these include (1) AKT-dependent phosphorylation of mouse double minute 2 protein (MDM2) resulting in the loss of activity of the pro-death mediator transformation-related protein 53 (TRP53; Mayo & Donner 2001); (2) phosphorylation (and inhibition) of glycogen synthase kinase 3 (GSK3; Fang et al. 2000); (3) phosphorylation and inhibition of the pro-apoptotic mediator Bcl-associated death promoter (BAD; Kulik & Weber 1998) (indirectly resulting in the inhibition of pro-apoptotic BCL2-associated X protein (BAX)); and (4) phosphorylation and inhibition of forkhead-related transcription factor (FKHR/DAF16; Nishikimi et al. 1999) (examples of the range of interactions can be found on the Science Database of cell signaling; http://stke.sciencemag.org/cm/). This multiplicity of downstream effectors provides a range of mechanisms through which ligand activation of PI3 kinase may induce cellular responses in the embryo.

The action of some of these effectors has been implicated in normal preimplantation embryo development. For instance, (1) AKT phosphorylates MDM2 and active MDM2 essential for preimplantation embryo (Montes de Oca Luna et al. 1995). MDM2 acts to maintain TRP53 in a latent state and latency of TRP53 is essential for embryo development and survival (Li et al. 2007a). (2) AKT results in activation Wnt/β-catenin pathway and activation of this pathway is essential for normal blastocyst implantation (Xie et al. 2008). (3) Phosphorylation and inhibition of the pro-apoptotic mediator BAD (Kulik & Weber 1998) (indirectly resulting in the inhibition of pro-apoptotic BAX/BCL2 antagonist/killer 1 (BAK)) and overexpression of BAX are associated with the loss of embryo viability (Moley et al. 1998, Chandrakanthan et al. 2006; Fig. 2).

Evidence for the activity of AKT in the early embryos infers that PDK is also active, although this has not been formally proved. PDK has a range of other downstream targets that includes P70S6 kinase, atypical protein kinase C, glucocortocoid- and serum-inducible kinases (Wick et al. 2002), and IB kinase complex (IKK; Tanaka et al. 2005). Activation of P70S6 kinase could explain the generalized trophic actions of many ligands on the early embryo. IKK may be an important target also, since its activation indirectly results in the activation of NF-B. NF-B is expressed in the preimplantation embryo and its inhibition from the earlier zygote stage blocks normal development (Nishikimi et al. 1999; Fig. 2). The PI3 kinase pathway may also play a role in mitigating the effects of genotoxic and non-genotoxic stressors on the early embryo, and this has been reviewed elsewhere (O'Neill 2008).

The net result of this complexity is that the generation of PIP3 amplifies the response of the cell to its ligand, generates a complex network of effectors in the responding cell, and as such provides for pleiotypic responses by embryo cells to discrete stimuli.

PIP3 degradation regulates cellular responses
The rate at which PIP3 is degraded is central to the strength and duration of any cellular response to a ligand. PIP3 is inactivated by selective phosphatases. Phosphatase and tensin homolog deleted on chromosome ten (PTEN; also known as mutated in multiple advanced cancers 1, MMAC1) reverses the action of PI3 kinase, converting PIP3 to PIP2 (Box 1; Fig. 1). Another phosphatase, SH2-containing inositol 5'-phosphatase (SHIP), is capable of converting PIP3 into PtdIns(3,4)P2 (Fruman et al. 1998, Cantley & Neel 1999).

Genetic deletion of these two major PIP3 phosphatases, PTEN (Cristofano et al. 1998) and SHIP (Helgason et al. 1998), results in markedly different phenotypes. Loss of PTEN results in early embryonic demise while SHIP^–/– embryos are viable. This indicates that PTEN activity is essential for normal embryo development but that is not the case for SHIP. Embryopathy in PTEN^–/– embryos occurs before day 7.5 of development, but detailed analysis of development has not been made. The persistence of PI3 kinase-mediated responses (such as elevated intracellular calcium concentration) that would be expected to occur in the absence of PTEN would be deleterious to the normal development of the embryo. Hence, an important role for PTEN in temporally limiting PI3 kinase-mediated responses in the early embryo is likely to be an important homeostatic mechanism.

It is thought that PTEN is constitutively expressed at a basal level in most cell types. Its acute regulation in response to ligands may have a role in fine-tuning cellular responses (Downes et al. 2007). PTEN is itself a target for phosphorylation. It is thought that phosphorylated serine and threonine residues present in the C-terminal tail interact with the basic regions in the phosphatase and C2 domain of PTEN and cause it to remain inactive. Dephosphorylation of the C-terminal tail residues results in an open conformation in the basic regions in PTEN, which can interact with anionic lipids, thus enhancing its association with the plasma membrane and orientation of the active site towards its lipid substrates (Downes et al. 2007).

To date, systematic analysis of the expression or activity of PTEN or SHIP in the preimplantation embryo has not been reported. The activity of these phosphatases and their influence on ligand-initiated, PIP3-mediated responses are priority areas for detailed study in the field.

	Conclusion

Top Abstract Introduction Synthesis of 3'... Evidence for a role... Action of embryotrophic ligands... Activation of PI3 kinase... Downstream effectors of PIP3... Conclusion Declaration of interest References

 
The manner of a given cell's response to ligand-mediated generation of PIP3 in the early embryo is governed by the receptor that is activated, the range of effectors that are expressed within the cell at the time of stimulation, the nature of the interactions (crosstalk) with other simultaneous signaling events occurring, and the rate of PIP3 degradation by PTEN. An understanding of how the array of potential responses in the embryo is coordinated in response to PIP3 is still in its infancy. As a minimum, this response will influence the embryo's capacity to survive and proliferate.

The developing evidence for the actions of ligand-mediated activation of PI3 kinase and the resulting generation of PIP3 implicate this signaling network as an important component of the regulation of the early embryo's homeostasis. A detailed characterization of the function of the signaling networks generated by PIP3 at each stage of embryo development will be an important step in understanding the autopoietic nature of early embryo growth and survival.

	Declaration of interest

Top Abstract Introduction Synthesis of 3'... Evidence for a role... Action of embryotrophic ligands... Activation of PI3 kinase... Downstream effectors of PIP3... Conclusion Declaration of interest References

 
The author declares that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Funding

 
This study was supported by grants from the Australian National Health and Medical Research Council.

Received March 10, 2008
First decision April 22, 2008
Accepted May 15, 2008

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Top Abstract Introduction Synthesis of 3'... Evidence for a role... Action of embryotrophic ligands... Activation of PI3 kinase... Downstream effectors of PIP3... Conclusion Declaration of interest References

 

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