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BOR - Papers in Press, published online ahead of print July 18, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.060129
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BIOLOGY OF REPRODUCTION 77, 813–821 (2007)
DOI: 10.1095/biolreprod.107.060129
© 2007 by the Society for the Study of Reproduction, Inc.

Direct Evidence for the Action of Phosphatidylinositol (3,4,5)-Trisphosphate-Mediated Signal Transduction in the 2-Cell Mouse Embryo1

Yan Li 3 4 5 6, Vashe Chandrakanthan 3 5 6, Margot L Day 5, and Chris O'Neill 2 4 5 6

Disciplines of Medicine4 and Physiology,5 University of Sydney, and Human Reproduction Unit,6 Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia

ABSTRACT

Paf (1-o-alkyl-2-acetyl-sn-gylcero-3-phosphocholine) is a putative autocrine survival factor for the preimplantation embryo. It acts to induce receptor-mediated calcium transients in the early embryo. Inhibitors of 1-o-phosphatidylinositol-3-kinase (PI3kinase), such as wortmannin and LY 294002, blocked these calcium transients, implicating the generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in autocrine signal transduction in the early embryo. Perfusion of the embryo cytoplasm with a blocking antibody to PIP3 inhibited paf-induced calcium transients and hyperpolarization of the membrane potential. Furthermore, direct infusion of PIP3 into the embryo induced a nifedipine (10 µmol/L)- and diltiazem (10 µmol/L)-sensitive calcium current in the 2-cell embryo. PIP3 acts as a docking site on membranes for proteins that contain pleckstrin homology domains, such as the thymoma viral proto-oncogene protein (AKT) and phospholipase C gamma. The 2-cell embryo expressed three genes for AKT (Akt 1–3) and two genes for phospholipase C gamma (Plcg1 and Plcg2), and we confirmed the expression of both AKT and phospholipase C gamma 1 by immunolocalization. Paf induced increased accumulation of serine 473-phosphorylated AKT in the region of the plasma membrane, consistent with its recruitment to membrane PIP3. Inhibitors of PI3kinase, such as LY294002, and of AKT, e.g., deguelin and AKT-inhibitor, reduced zygote development in a dose-dependent manner, and this inhibition was partially reversed by the addition of paf to the culture medium. These results provide the first direct evidence that PIP3 and its responsive signaling pathways act in the 2-cell embryo. Since signal transduction via PI3kinase has important roles in governing the cell survival pathways, these results support the hypothesis that autocrine embryotropins, such as paf, act as survival factors.

calcium, calcium channel, developmental biology, early development, embryo, membrane potential

INTRODUCTION

A current paradigm in cell biology is that each cell type requires exposure to a range of survival factors to ensure its viability. It is proposed that cells constitutively express death programs, and that the role of survival factors is to maintain these programs in a latent state [1, 2]. In the absence of survival signaling, cell death is the default response [3]. In somatic cells, survival signals are typically generated by neighboring cells within tissues, which act in a paracrine fashion [1]. As a result, survival factors act to ensure that cells are constrained to survive only in a location (or environment) in which they are exposed to their restricted repertoire of survival factors. Teleologically, it is often argued that survival factors act as a mechanism that limits metastasis or ectopic growth of cells. This argument is supported by the observation that many tumor cells have a reduced requirement for exogenous survival factors. During development, survival factors may have a role in tissue modeling [4].

The preimplantation mammalian embryo shows apparent autonomy from survival factors. This is most apparent during the culture of embryos in vitro, where embryos develop in entirely defined media and in the absence of hormones or growth factors. This autonomy is taken as evidence that the early embryo is exempt from the requirement for survival factor action displayed by somatic cells [3]. However, a range of trophic ligands has been shown to be expressed by the early embryo [5–7]. Several of these ligands are produced by the embryo and act upon their respective receptors, which are also expressed by the embryo, and are thus classed as autocrine factors. It has been proposed that these autocrine ligands act as survival factors [8, 9].

The actions of autocrine ligands may be largely overlapping or redundant [9]. These factors include paf (1-o-alkyl-2-acetyl-sn-gylcero-3-phosphocholine) [10], insulin-like growth factor 1, insulin-like growth factor 2 [11], and transforming growth factor (TGF) {alpha} [12]. It is also likely that a range of paracrine and endocrine factors exerts similar actions on the embryo; examples include insulin [13] and colony stimulating factor 2 (granulocyte-macrophage) [14]. Redundancy among these factors is consistent with observations of somatic cells, in which survival signaling is conferred by several paracrine trophic factors. Where such redundant action has an autocrine basis, it provides a particular challenge to investigators, in that it is not readily amenable to classic ablation techniques. To understand better the nature of autocrine trophic actions in the early embryo, we have investigated the nature of autocrine signal transduction.

Survival factors in many cell types act via receptors that activate 1-o-phosphatidylinositol-3-kinase (PI3kinase) [15]. PI3kinases, which are proto-oncogenes, induce the phosphorylation of membrane inositol phospholipids, resulting in the formation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) [16, 17]. PIP3 acts as a docking site for a range of proteins that contain the pleckstrin homology (PH) domain [16]. The generation of PIP3 results in translocation of PH domain-containing proteins to the membrane [16]. This translocation commonly results in their functional activation. An important protein that acts in this manner is thymoma viral proto-oncogene (AKT), which is phosphorylated (and activated) upon membrane translocation [18–20]. Activated AKT exerts many important functions related to cell survival, proliferation, and differentiation [18].

A role for PI3kinase in the normal development of the preimplantation embryo was discovered following the observation that two pharmacologically distinct inhibitors of PI3kinase, wortmannin and LY 294002, could cause failure of normal preimplantation development of mouse zygotes in vitro [9]. Furthermore, it has been shown that several different forms of PI3kinase are expressed in the early embryo [9]. The presence of multiple isoforms of the enzyme excludes convenient genetic ablation analysis of the role of this enzyme activity.

Arguably, the first of the trophic ligands to act on the embryo is paf [7]. Paf can be extracted from culture media conditioned by the embryos of various mammalian species [21–23], while the addition of paf to culture media enhances embryo metabolism [10], growth rates [24–26], and viability [27, 28]. The embryo expresses a paf receptor (Ptafr) [29, 30], and in other cell types, this receptor is known to activate PI3kinase [31]. The action of embryo-derived paf during the zygote and 2-cell stages is required for normal development of the mouse embryo in vitro [7, 8, 32]. Paf induces periodic transient increases in the intracellular calcium concentration ([Ca2+]i) in the mouse zygote and 2-cell embryo [33], and these fluxes are necessary for normal embryo development in vitro [9, 34]. These transients are mediated by Ptafr, are pertussis toxin-sensitive [9, 33], and are blocked by PI3kinase blockers [9]. The Paf-induced [Ca2+]i transient requires the action of phospholipase C (PLC)-induced release of internal calcium stores [33], as well as an influx of external calcium through a dihydropyridine-sensitive calcium channel [33, 34]. PLC (isoforms G [35] and D1 [36]) possess PH domains and are activated in response to PI3kinase, and dihydropyridine-sensitive calcium channels may be regulated in part by PI3kinase [37].

Wortmannin and LY 294002 are recognized as selective inhibitors of PI3kinase over a narrow dose range, yet they can also block the intrinsic serine kinase activity of PI3kinase [38]. Thus, the reliance on these pharmacological inhibitors to demonstrate a role for PI3kinase does not exclude the possibility that this enzyme is primarily acting as a protein serine kinase. Thus, current evidence does not provide unequivocal proof that putative survival factors, such as paf, act through the generation of PIP3. Consequently, it cannot currently be inferred that activation of PH domain-containing proteins, including the plethora of such proteins that govern cell survival, results from the actions of these trophic ligands.

The aims of the current study were to 1) investigate whether paf-induced signal transduction requires the generation of PIP3 in the 2-cell embryo by examining the effects of a blocking antibody to PIP3; 2) determine whether the direct infusion of PIP3 upon calcium signaling in the early embryo induces signaling events; and 3) assess whether a PH domain-containing protein (AKT) translocates to the membrane in response to paf. Our results provide direct evidence that the generation of PIP3 is a component of the signal transduction induced by paf in the 2-cell mouse embryo, and thus support the conclusion that paf acts as an autocrine survival factor for the 2-cell embryo.

MATERIALS AND METHODS

Animals

The use of animals was in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose and was approved by the Institutional Animal Care and Ethics Committee. The following mouse strains were used in the experiments: C57BL/6 (B6); C57BL/6 x CBA/He (B6CBF1); and Ptafr–/- (paf-receptor null; extensively backcrossed with B6). The Ptafr–/- genotype of all the knockout mice was confirmed by PCR.

Female mice, 6 weeks old, were superovulated by i.p. injection of 5 IU eCG (Folligon; Intervet International, Boxmeer, The Netherlands) followed 48 h later by 5 IU hCG (Chorulon; Intervet). Females were then paired with males of proven fertility. Day 0.5 of pregnancy was confirmed by the presence of a copulation plug.

Embryo Collection

All components of media were tissue culture grade (Sigma Chemical Co., St. Louis, MO). Unless otherwise stated, all media were supplemented with 0.3% (w/v) BSA (Fraction V; CSL Ltd., Melbourne, VIC, Australia).

The 2-cell embryos were flushed from the reproductive tract using Hepes-buffered modified human tube fluid medium (Hepes-modHTF) [8], which contained (in mmol/L) NaCl (101.6), KCl (4.7), MgSO4 (0.2), KH2PO4 (0.4), sodium lactate (21.4), glutamine (1.0), sodium pyruvate (0.3), glucose (2.8), CaCl2 (2.0), NaHCO3 (4.2), and Hepes (20.4) (pH 7.35 and 285 mOsm/kg).

Immunofluorescence

Embryos were washed three times in PBS that contained 0.1% BSA, 0.1% Tween-20, and 0.2% (w/v) sodium azide (washing buffer), fixed with 2% (w/v) paraformaldehyde (Sigma) for 30 min, and then permeabilized with 2% paraformaldehyde plus 0.3% Tween-20 (Sigma) at room temperature for 30 min. Embryos were washed three times in washing buffer, blocked in 2% BSA and 30% serum for 3 h, and stained overnight at 4°C with the following primary antibodies: a) rabbit anti-PLCG1 polyclonal antibody raised against amino acids 530–850 of rat origin (sc-426, final concentration of 10 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA); b) anti-phospho(Ser473)-AKT (193H12) rabbit monoclonal antibody (final concentration of 2 µg/ml; Cell Signaling Technology, Danvers, MA); or c) an equivalent concentration of nonimmune immunoglobulin. Embryos were washed thoroughly three times for 10 min each in 1x PBS and incubated with a 1:200 dilution of goat anti-rabbit FITC-conjugated IgG secondary antibody (Sigma).

Embryos were collected fresh from the reproductive tract. The embryos were washed three times in cold PBS (Ca2+- and Mg2+-free Dulbecco PBS [Sigma]) and then transferred in a minimal volume to 30 µl of PCR buffer (50 mM KCl, 15 mM Tris-HCl [pH 8.0]) in diethyl pyrocarbonate (DEPC; Sigma)-treated MilliQ water that contained 1 IU of RNase inhibitor (Applied Biosystems, Foster City, CA). The embryos were lyzed by three cycles of freezing in liquid nitrogen and thawing (with vortexing), and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR). Reverse transcription was performed in 12.5 U of murine leukemia virus reverse transcriptase (MuLV), 1 U RNase inhibitor, 4 mM MgCl2, 50 mM KCl, 15 mM Tris-HCl (pH 8.0), 0.5 mM dNTPs (Applied Biosystems), 1.5 µM allele-specific reverse primer. The reactions were incubated for 10 min at room temperature, 30 min at 42°C, and 2 min at 99°C. Two negative controls were included: without reverse transcriptase enzyme, and without template, to test for extraneous RNA and DNA contamination, respectively. The PCR mixture contained 3 µl cDNA template, 1.5 µl Amplitaq Gold DNA polymerase, 4 mM MgCl2, 50 mM KCl, 15 mM Tris-HCl (pH 8.0), .5 mM dNTPs, 5% dimethylsulfoxide (DMSO; Sigma), and 1.5 µM each of the gene-specific primers. The reactions were incubated for 10 min at 94°C, followed by 40 cycles of 15 s at 94°C and 1 min at 58°C in a Corbett Thermal Reactor. The PCR products were analyzed by electrophoresis on a 2% (w/v) agarose gel that was stained with ethidium bromide, to visualize the products on a UV transilluminator. The DNA fragments were verified by size and the products were extracted and sequenced (SUPAMAC, Redfern, NSW, Australia), to confirm they were from the target genes.

The primers were designed using the Prima software (ANGIS, Sydney, NSW, Australia) and purchased from Sigma-Genosys (Sigma). Actb was used as a positive control for all tissue and embryo samples. The sequences of the forward and reverse primers (and the product sizes) were as follows: for Akt1 (accession number NM009652), 5'-CGGATACCATGAACGACGTAG-3' and 5'-GCAGGCAGCGGATGATAAAG-3' (243 bp); Akt2 (XM122192), 5'-ACCTTTGTCATACGCTGCC-3' and 5'-CGAACCAAAATGACCTTGCC-3' (296 bp); for Akt3 (NM_011785), 5'-TGCCTTCTCTCGAACCAAAA-3' and 5'-TGCCTTCTCTCGAACCAAAA-3' (106 bp); for Plcb1 (AF498249), 5'-GCACACGACCAAGTACAACGAG-3' and 5'-ATTTCTGCATCCAGGGCAGC-3' (162 bp); for Plcb2 (XM_110361) 5'-GGACAAGCAGTTCAACCCCTTC-3' and 5'-GGCCAAACAGTTCCACTTCCAC-3' (137 bp); for Plcg1 (XM_130636), 5'-TGGAGAGGAGGAAGAAGATCGC-3' and 5'-ACATGTCCCGGTAACAAGCAC-3' (114 bp); for Plcg2 (BC023877) 5'-AGACGAAGGCAGACAGCATTG-3' and 5'-GCCCATTGAGCGAAAACAGC-3' (219 bp); and for Actb (MMACTBR), 5'-CGTGGGCCGCCCTAGGCACCA-3' and 5'-TTGGCCTTAGGGTTCAGGGGG-3' (243 bp).

Patch-Clamp Methods

Standard whole-cell patch-clamp techniques were used to study paf-induced currents and changes in the membrane potential (Em) of 2-cell embryos. Immediately before their use in patch-clamp or imaging experiments, groups of 3–5 embryos were treated with 0.5% (w/v) pronase at 37°C for 3 min, to remove the zona pellucida, and then washed in Hepes-modHTF. All patch-clamp studies were performed on zona pellucida-free embryos. The List EPC-7 patch-clamp amplifier (List, Darmstadt, Germany) was used in all patch-clamping experiments. Currents were low-pass-filtered, sampled, and digitized at 0.2 kHz with a MacLab-4 data acquisition interface (AD Instruments, Sydney, NSW, Australia). Patch-clamp pipettes were manufactured from borosilicate tubes (Modulohm, Herley, Denmark). All patch-clamp experiments were performed at 36–37°C by constant perfusion of the bath solution at a rate of 1 ml/min.

The Em was recorded using a pipette solution that contained (in mmol/L): KCl (140), MgCl2 (1), Hepes (10), glucose (10), EGTA (0.5), EDTA (0.01) (pH 7.2 and 283 mOsM/kg). Changes in the membrane ion currents were assessed by voltage-clamping at 0 mV immediately after a whole-cell patch was obtained. Whole-cell currents were recorded using a pipette solution in which KCl was replaced by N-methyl-D-glutamine (NMDG) glutamate and contained (in mmol/L) NMDG (115), NaCl (25), MgCl2 (1), Hepes (10), glucose (10), EGTA (0.5), and EDTA (0.01), adjusted to pH 7.2 with glutamic acid. Nystatin (240 µg/ml; Sigma) was added to the pipette solution to perforate the embryo plasma membrane. The bath solution was Hepes-modHTF; however, to provide a greater driving force for calcium currents, the concentration of calcium was increased to 3 mol/L in some experiments.

Calcium Imaging

Zona-free 2-cell embryos were prepared as described above and loaded with 2 µM Fluo-3 AM (Molecular Probes, Eugene, OR) in Hepes-modHTF that contained BSA and 0.02% (w/v) pluronic acid, for 15 min at 37°C, or with 2 µM Fura-2 AM (Molecular Probes) in BSA-free perfusion medium for 30 min. After loading, embryos were washed three times in the appropriate Hepes-modHTF and then treated with recombinant plasma-type paf acetylhydrolase (175 µg/ml; a gift from ICOS Inc., Bothell, WA) at 37°C for 15 min, to degrade endogenous stores of embryo-derived paf [33]. All operations were protected from light. Fluo-3 was excited using a 480-nm filter and detected using a 535/540-nm filter, and fluorescence intensity was recorded. Ratiometric imaging of Fura-2 fluorescence was performed at excitation wavelengths of 340 nm and 380 nm.

Treatments

Paf (equal mixture of 1-o-octadecyl/hexadecyl-2-acetyl-sn-glyceryl-3-phosphocholine; Sigma) was prepared as a 1 mg/ml stock solution in chloroform). Aliquots were removed to a siliconized glass test tube, reduced to dryness under a stream of N2, and dissolved in perfusion medium to the desired concentration. Paf was dissolved in Hepes-modHTF perfusion medium that contained 3 mg/ml BSA at a final concentration of 372 nmol/L. PIP3 (1,2-dipalmitoyl, sodium salt; Cayman Chemical, Ann Arbor, MI) was prepared as a 0.5-mM stock in the pipette solution and stored at –70°C. A final concentration of 4 µM PIP3 was prepared in the NMDG glutamate pipette solution and used within 1 h of preparation. An antibody that blocks PIP3 action (mouse IgM monoclonal RC6F8) has been validated [37] and was purchased from Molecular Probes. It was supplied as a 1 mg/ml stock in PBS that contained 0.1% (w/v) BSA. A final concentration of 10 µg anti-PIP3 antibody/ml was used in the pipette solution. In some experiments, the antibody was boiled prior to use. Medium that contained 1 µmol/L free Ca2+ was made by adding 2.14 mM EGTA to Hepes-modHTF. The calcium channel blockers diltiazem and nifedipine (Calbiochem, Alexandria, NSW, Australia) were prepared in perfusion medium. The inhibitors LY294002 (Calbiochem), AKT inhibitor (1L-6-Hydroxymethyl-chiro-inositol 2-[(R)-2-O-methyl-3-O-octadecylcarbonate]; Calbiochem), and deguelin ((7aS, 13aS)-13,13a-dihydro-9, 10-dimethoxy-3, 3-dimethyl-3'H-bis [1] benzopyrano [3,4-b:6',5'-e]pyran-7(7aH)-one; Sigma) were initially prepared as 2000-fold concentrated stocks in DMSO and subsequently diluted to the working concentrations in perfusion medium or culture medium, as required. In all experiments in which DMSO was used as the solvent, the control medium contained the same concentration of DMSO. In the calcium imaging studies, embryos were pretreated with these agents for 20 min and also treated with the antagonist throughout the paf challenge.

Statistical Analysis

The data are expressed as the mean ± SEM. Differences between treatments were analyzed by the Student t-test. Differences in the proportion of embryos that displayed responses were compared using the chi-square test.

RESULTS

The measurement of [Ca2+]i was performed simultaneously with the measurement of the membrane potential (Em) or ion current activity, by performing whole-cell patch clamp analysis of 2-cell embryos that were preloaded with the calcium-sensitive dye Fluo-3. This confirmed that exposure of the 2-cell mouse embryo to paf in vitro induced a characteristic calcium transient, and showed that this was accompanied by a transient hyperpolarization of the Em (Fig. 1, Ai, B, and C). Infusion of a blocking antibody to PIP3 into the cytoplasm through the patch pipette reduced both the paf-induced [Ca2+]i transient (P < 0.00001) and hyperpolarization of Em (P < 0.003) in the 2-cell embryos (Fig. 1, Aii, B, and C). In the small proportion of embryos treated with the PIP3 blocking antibody that still elicited a response, the response was significantly delayed compared with the controls (P < 00001) (Fig. 1D). After the PIP3 blocking antibody was boiled it lost the capacity to block the responses of the embryo to paf, thereby confirming the specificity of the action of the antibody infusion (Fig. 1, Aiii, B–D).


Figure 01
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  		FIG. 1 The effect of PIP3 blocking antibody on the responses of 2-cell embryos to paf. Embryos (B6) were perfused with a PIP3 blocking antibody and challenged with exogenous paf. Simultaneous measurements of the relative changes in [Ca2+]i and Em were performed. A) Representative traces using (i) KCl pipette solution (n = 12); (ii) pipette solution containing 10 µg anti-PIP3 antibody/ml (n = 8); or (iii) pipette solution containing boiled anti-PIP3 antibody (n = 5). B) The size of the Em hyperpolarization. C) The relative change in [Ca2+]i. D) The time to onset of [Ca2+]i response after paf challenge. The values shown are means ± SEM. A t-test was used to compare the differences between the treatments.

Direct infusion of PIP3 into the cytoplasm of 2-cell embryos via the patch pipette induced a net inward cationic current (Fig. 2, A and B). This current did not occur either after infusion of vehicle alone or when the calcium concentration in the medium ([Ca2+]o) was reduced to 1 µM (P < 0.001) (Fig. 2, A and B). Furthermore, this current was reduced to control levels when the calcium channel blockers nifedipine (10 µM) or diltiazem (10 µM) were present in the bath solution (Fig. 2C). The observations that a PIP3 blocking antibody inhibits paf-induced signaling, while PIP3 infusion induces activation of a calcium current provides direct evidence for the role of PIP3 in the signal transduction induced by paf in the 2-cell embryo.


Figure 02
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  		FIG. 2 The effect of PIP3 on inward cation current activity within 2-cell embryos. Conventional whole-cell patch-clamps (with NMDG glutamate pipette solution) were applied to B6 2-cell embryos. The Em was voltage-clamped at 0 mV. A) Representative traces of membrane currents obtained for embryos treated with PIP3 or vehicle in bath solution that contained 2.04 mM or 0.001 mM [Ca2+]o. B) The average amplitude (± SEM) of the PIP3-induced inward current for the three treatments described in A. C) The effects of calcium channel blockers (nifedipine or diltiazem) on the PIP3-induced inward current in 2-cell embryos in the presence of 3 mM [Ca2+]o.

Treatment of embryos with the PI3kinase inhibitor LY294002 (P < 0.005) caused dose-dependent inhibition of preimplantation embryo development to the blastocyst stage (Fig. 3A). Inhibition of AKT with deguelin also caused dose-dependent inhibition of embryo development (P < 0.005) (Fig. 3B). Supplementing the culture media with paf significantly attenuated the inhibitory effects of LY294002 (P < 0.05) and deguelin (P < 0.05) (Fig. 3, A and B). There was also a significant interaction effect between paf and LY294002 treatment (P < 0.05), but not between paf and deguelin treatment (P > 0.05). Both inhibitors caused a significant dose-dependent adverse effect on the total number of cells within each blastocyst (LY294002, P < 0.005; deguelin, P < 0.05) (Fig. 3, C and D, respectively). A second inhibitor of AKT (AKT-inhibitor) also caused significant dose-dependent inhibition of zygote development to the blastocyst stage (P < 0.001) and reduced the number of cells in the resultant blastocysts (P < 0.001) (Fig. 4A). Of interest was the observation that AKT-inhibitor did not affect the [Ca2+]i transient induced by paf, which suggests that the activation of the calcium transient is downstream of PI3kinase, but not AKT activation (Fig. 4B).


Figure 03
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  		FIG. 3 The effect of LY294002 or deguelin on the proportion of zygotes that develop to the blastocyst stage and the interaction of paf supplementation of the media. F1 zygotes were cultured individually in 10-µl drops of media with (pafr) and without (Media) paf for 96 h. A) The proportion of zygotes that developed to the blastocyst stage when cultured with a range of doses of LY294002. B) The proportion of zygotes that developed to the blastocyst stage when cultured with deguelin. C) The number of cells in embryos that did develop to blastocysts after culture in LY294002. D) The number of cells in deguelin-treated embryos that developed to blastocysts. The results shown are from four separate replicates. Statistically significant effects are described in the text.


Figure 04
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  		FIG. 4 A) The effect of AKT-inhibitor on the development of zygotes in vitro. Zygotes (F1) were cultured individually in 10-µl drops of media for 96 h. The proportion of zygotes that developed to normal blastocysts after 96 h culture and the number of cells per embryo (mean ± SD) are shown. The results shown are from three separate replicates. Statistically significant effects are described in the text. B) The effect of AKT-inhibitor on paf-induced Ca2+ transients in F1 2-cell embryos. Traces show changes in [Ca2+]i in F1 2-cell embryos in response to paf (37 nmol/L) in the presence of AKT-inhibitor (0, 3, or 15 µmol/L). The traces are the typical responses of the number of embryos shown in brackets. The results shown are from five separate experimental replicates.

AKT and PLCG both contain PH domains and can be activated upon binding to PIP3. At least three genes code for Akt, and the mRNAs for Akt1–3 were detected within the 2-cell embryo (Fig. 5, A and B). The two PLCG genes (Plcg1 and Plcg2) were also expressed in the 2-cell embryo (Fig. 5C), as were two forms of PLCB (Plcb1 and Plcb2) (Fig. 5D). PLCG1 protein was expressed in the 2-cell embryo (Fig. 5E1), confirming an earlier observation [39]. Staining occurred throughout the cytoplasm, and in about 20% of embryos, there were obvious foci of increased staining at sites in the region of the plasma membrane. There was no significant staining in the nonimmune controls (Fig. 5E2).


Figure 05
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  		FIG. 5 Expression of mRNAs for AKT and PLC in F1 2-cell embryos. RT-PCR was performed with specific primers to detect the gene products. In all the gels, lanes 1, 2, and 3 are reverse-transcriptase omitted, RNA omitted, and internal control (Actb, 243 bp), respectively. A) Lane 4, Akt1 (243 bp); and lane 5, Akt2 (296 bp). B) Lane 4, Akt3 (106 bp). C) Lane 4, Plcb2 (219 bp); and lane 5, Plcb2 (137 bp); D) Lane 4, Plcg2 (219 bp); and lane 5, Plcg1 (114 bp). E) 1) Expression and immunolocalization of PLCG1 in a 2-cell embryo, and 2) nonimmune immunoglobin control; F) Expression and localization of pAKT in 2-cell embryos exposed to control media or paf. Immunofluorescence staining of Ser473 phospho-AKT in 2-cell B6 embryos (Ptafr+/+) or embryos that lack the paf-receptor gene (Ptafr–/–). Embryos were cultured in the presence (paf) or absence (Media) of 37 nM paf for 0, 1, or 2 h. Images of duplicate embryos for each treatment are shown. Embryos were exposed to antibody to pAKT (1–20), secondary antibody only (21, 22) or nonimmune serum (23, 24). Images were subjected to deconvolution and are representative of three replicates. Original magnification x225.

Activation of AKT is accompanied by its phosphorylation at Ser473 (pAKT) [40]. Using activation state-specific antibodies to pAKT, staining was detected throughout the cytoplasm, with some accumulation in the perinuclear regions of some cells (Fig. 5F, lanes 1–4). Embryos cultured for 1 h (Fig. 5F, lanes 5–8) showed similar staining to those exposed to paf (Fig. 5F, lanes 9–12) (at 15 min or 30 min, data not shown). However, after 2 h in culture (Fig. 5F, lanes 13–16), exposure to paf increased the staining in the cortical region of the blastomeres (Fig. 5F, lanes 17 and 18), suggesting translocation and phosphorylation of AKT to the cell membrane. The increased cortical localization in response to paf was dependent upon the action of Ptafr, since it was not observed in embryos that lacked the receptor (Ptafr–/–) (Fig. 5F, lanes 19 and 20). Paf treatment did not cause an obvious increase in the total pAKT staining in the embryo. Negligible staining was observed for either the secondary antibody alone (Fig. 5F, lanes 21 and 22) or the nonimmune control (Fig. 5F, lanes 23 and 24).

DISCUSSION

Improvements in the development and survival of embryos cultured in groups are commonly taken as evidence for the action of autocrine factors. This effect has been best demonstrated in mice, but has also been reported in cattle [41, 42], sheep [43], cats [44], and humans [45, 46]. In the human, improved development occurs if embryos are cultured in groups from the zygote stage [45, 46], but not apparently if the coculture commences at Day 3 [47]. This finding is consistent with the observation that trophic support is best applied prior to the completion of the 2-cell stage of development in the mouse [32]. In cattle, PI3kinase activity is required to protect early embryos from stress-induced apoptosis [48]. We are unaware of any other reports of a putative action of PI3kinase in the early embryos of other mammalian species, although insulin signaling, which is mediated by DAF-2 through the AGE-1 phosphatidylinositol-3-OH kinase, regulates embryonic development in Caenorhabditis elegans [49], and pharmacologic inhibition of PI3kinase blocks early embryo development in sea urchins [50].

Pharmacologic inhibitors are powerful experimental tools, yet conclusions must be constrained by their specificities of action. PI3kinase inhibitors are also known to inhibit the intrinsic serine kinase activity of the enzyme [38]. The present study confirms that paf induces a [Ca2+]i transient and shows that this is accompanied by hyperpolarization of the Em in the 2-cell embryo. We show for the first time that direct inhibition of the action of PIP3 using a blocking antibody inhibits both responses to paf in the 2-cell embryo. We further demonstrate that direct infusion of PIP3 into the embryo activates a diltiazem- and nifedipine-sensitive calcium current, and thus mimics some of the actions of paf. The blocking antibody used in the present study has previously been validated as being effective in blocking PIP3-mediated calcium channel activity [37]. The results of the present study provide the first direct evidence for PIP3-mediated signal transduction in the 2-cell mammalian embryo, and thus directly implicate PI3kinase activity in signal transduction in these embryos.

The generation of PIP3 provides docking sites for a host of proteins that contain the PH domain. Many of these proteins are central to the regulation of cellular survival and proliferation (reviewed in [20]). This capacity of PIP3 to dock and activate such a range of proteins accounts for the pleiotypic actions of PI3kinase. We show here that the 2-cell embryo expresses five genes that code for proteins with PH domains (Akt1–3 and Plcg1 and 2). Many of the cell survival and proliferative functions of PI3kinase are exerted through the activation of AKT [16, 18, 19]. The interaction of the AKT PH domain with PIP3 results in the translocation of AKT to the plasma membrane. It is generally considered that phosphorylation of AKT occurs as a consequence of translocation. Phosphorylation occurs at least at two sites, one of which lies in the activation loop (Thr308) and the other in the carboxy-terminal tail (Ser473) [51]. Phosphorylation at Ser473 may precede and facilitate phosphorylation at Thr308 ([51] and references therein). The kinase(s) responsible for Ser473 phosphorylation have not been identified unequivocally, but may include the mammalian target of rapamycin (mTOR), Gbl, and rictor [51]. Our present study confirms the expression of pAKT [52], and shows for the first time that paf induces the localization of pAKT to the region of the plasma membrane. This relocalization infers (without direct proof) the binding of AKT to membrane PIP3. This translocation occurred after a surprisingly long delay (~2 h). Although there was increased staining in the membrane, subjectively, there did not appear to be a marked increase in the overall levels of pAKT staining. During 2 h of exposure to paf, at least two [Ca2+]i transients would be expected to occur [33]. The results suggest that either more than one transient is required to induce the translocation in vitro or that other functional units, which require time for their recruitment, are also required for the membrane localization of AKT in the embryo. The nature of AKT activation in response to paf requires further investigation.

Multiple forms of Akt were expressed in the early embryo. The actions of the three known AKTs overlap to a considerable degree (reviewed in [53]). Genetic deletion of individual Akt genes results in modest phenotypes [54–56]. The Akt1–/–Akt2–/– compound mutants die at or soon after birth [57]. Akt1–/–Akt3–/– double-mutant mice die in utero [58]. Since the phenotypes of these compound mutants are more severe than the sum of the single null phenotypes, a high degree of functional compensation among the three Akt genes is indicated. Triple mutants have not been reported. Given the considerable overlap of AKT function and the lethality of the double mutant, the selective pharmacologic inhibition of AKT is currently the best tool for investigating AKT function in the early embryo. Deguelin is a natural product isolated from Mundulea sericea (Leguminosae) [59]. This inhibitor selectively inhibits all AKT isoforms (IC50 of 10 nM) [60, 61]. Deguelin (at concentrations >100 nM) nonselectively inhibits PI3kinase but not mitogen-activated protein kinase (MAPK) 3, MAPK 1/2 or MAPK 8 [62]. AKT-inhibitor is a phosphatidylinositol ether analogue. It selectively inhibits all AKT isoforms (IC50 of 5 µM), while it is only a weak inhibitor of PI3kinase (IC50 of 83 µM) [63, 64]. The similar actions on the embryo of these two structurally and pharmacologically distinct AKT antagonists suggest that inhibition of AKT is not consistent with normal embryo survival in vitro. The similar inhibitory profiles of putative PI3kinase and AKT inhibitors on zygote development are consistent with these agents acting within the same signaling pathway in the early embryo. The observation that paf-induced [Ca2+]i signaling was not blocked by inhibition of AKT but was blocked by inhibition of PI3kinase [33] argues for the independence of the calcium signal from AKT action. These results also indicate that AKT-inhibitor at the concentrations used was not acting by inhibiting PI3kinase.

There are many important regulatory pathways downstream of AKT [15, 19]. One important downstream target of AKT is transformed mouse 3T3 cell double minute 2 (MDM2) [65]. MDM2 acts as an E3 ubiquitin ligase that maintains transformation related protein 53 (TRP53) in a latent state by inducing its rapid degradation [66, 67]. A deficiency in signaling via PI3kinase can lead to loss of MDM2 activity and consequent accumulation of TRP53. TRP53 is a transcription factor that can produce a transcriptome that leads to either cell death or decreased cell cycle progression [68]. It has recently been shown that the culture of zygotes in vitro, where survival factor signaling is thought to be limiting [8], results in increased TRP53 expression [69] and transcriptional activity [70]. This causes a marked loss of viability of the resulting embryos [69]. It remains to be determined whether the increase in TRP53 is due to limited AKT function during culture in vitro. Baculoviral IAP repeat-containing 5 protein (survivin) is a member of the inhibitor of apoptosis protein family and is expressed throughout the preimplantation stage of development [71]. Its expression is upregulated by TGF {alpha}, a putative autocrine embryotrophin. This upregulation is blocked by the two PI 3kinase inhibitors [71], and this ligand-induced signaling also counteracts apoptosis induced by exogenous TNF [72].

PLCG1 is expressed in 2-cell embryos, and selective pharmacologic inhibition of PLC inhibits paf-induced calcium transients [33]. Dihydropyridine-sensitive calcium channels are known to be activated in some circumstances by PI3kinase-dependent mechanisms [37]. The present study provides evidence for both activities in response to paf action on 2-cell embryos. It is known that the calcium transients that occur in response to paf action depend upon both the influx of external calcium through a dihydropyridine-sensitive calcium channel [33, 34] and the PLC/inositol trisphosphate-dependent release of internal calcium stores [33]. The present study shows that the activation of PI3kinase by paf provides a mechanism that may link the activation of these two sources of calcium. The calcium transients induced by paf are apparently important for normal embryonic survival in vitro, since buffering these transients compromises normal development [33]. Calmodulin is an important intracellular receptor for increased calcium, and it is known that normal calmodulin function is essential for normal early embryonic development [73]. Downstream of calcium/calmodulin signaling is the cAMP responsive element-binding protein (CREB) family of transcription factors [74], which is also essential for normal preimplantation development [75, 76].

The present study shows that paf induces signal transduction in the 2-cell embryo in a PIP3-dependent fashion. This provides the first direct evidence for PIP3-mediated signal transduction in the early embryo. The results support the conclusion that survival factors, such as paf, act via PI3kinase to activate prosurvival signaling pathways in the early embryo, and thus provide one mechanism for the developmental autonomy of the preimplantation mammalian embryo.

ACKNOWLEDGMENTS

We thank ICOS Corporporation for the gift of Paf acetylhydrolase, and the staff of Gore Hill Research Laboratories and Sydney University Animal House for the care and supply of animals.

FOOTNOTES

3These authors contributed equally to this work. Back

1Supported by grants from the NHMRC (to C.O.N) and the ARC (to M.L.D.). Y.L. and V.C. were supported by a University of Sydney Postgraduate Studentship and Australian Postgraduate Scholarship, respectively. Back

Correspondence: 2Chris O'Neill, Human Reproduction Unit, Royal North Shore Hospital, University of Sydney, St Leonards, NSW 2065, Australia. FAX: 61 2 9926 6343; e-mail: chriso{at}med.usyd.edu.au

Received: 14 January 2007.

First decision: 8 February 2007.

Accepted: 17 July 2007.

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