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A universal response to cellular stress is the expression of transformation-related protein 53 (TRP53). This transcription factor reduces cell proliferation and/or survival and is classed as a tumour suppressor protein. Several stresses (including culture) cause increased TRP53 expression in blastocysts and their reduced long-term developmental potential. This study shows that culture from the zygote stage (but not the 2-cell stage) reduced the development of C57BL6 inbred (but not hybrid) strain mouse embryos. Reduced viability was TRP53 dependent, being partially reversed by a TRP53 inhibitor (Pifithrin-alpha). However, the presence of culture did not cause an increase in Trp53 mRNA levels (levels were reduced following culture, P < 0.001). Transformed mouse 3T3 cell double minute 2 (MDM2) causes the ubiquitination and degradation of TRP53. MDM2 activation is accompanied by phosphorylation of Ser-166, and this is commonly catalyzed by the phosphatidylinositol-3 kinase and RAC-alpha serine/threonine-protein kinase (AKT) signaling pathway. Paf is an autocrine embryotrophin that activates the phosphatidylinositol-3 kinase/AKT pathway. High levels of TRP53 expression occurred following the culture of zygotes lacking the Paf receptor (Ptafr−/−) and following inhibition of phosphatidylinositol-3 kinase or AKT. Inhibition of MDM2 caused a Trp53-dependent reduction in zygote development. Inbred strain embryos cultured from the zygote stage expressed less phosphorylated MDM2 than similar embryos collected from the uterus. The addition of Paf to the media caused increased phosphorylation of MDM2, and this was blocked by inhibitors of phosphatidylinositol-3 kinase and AKT. The study identifies trophic ligand signaling via the activation of phosphatidylinositol-3 kinase and AKT as a mechanism resulting in the activation of MDM2.
Early embryos develop in an apparently autonomous manner from the time of fertilization until at least the blastocyst stage of development. This involves several rounds of mitoses and the first stage of cellular differentiation. During this phase of development, embryos seem to be particularly susceptible to a range of exogenous stressors. One example of this is the reduction in the viability of many embryos following their production by fertilization in vitro or when they are subjected to prolonged culture in vitro from the zygote stage. The variable loss of viability of embryos under such conditions is thought to be primarily a response of the embryo to a range of stressors that they may be exposed to in vitro. These stressors may include growth and survival factor deprivation [1, 2], metabolic and substrate imbalances [3, 4], oxidative stress , and osmotic and shear stresses , and may also involve gross or minor genetic  and epigenetic defects .
In somatic cells, all such stresses are capable of activating the transformation-related protein 53 (TRP53) stress response pathway . TRP53 is a transcription factor that can either reduce cycle-cell progression by, for example, the induction of cyclin-dependent kinase inhibitor 1A or induce apoptosis by, for example, the synthesis of pro-apoptotic mediators, such as Bcl2-associated X protein (BAX). Increased expression of TRP53 is an important mediator of the loss of embryo viability following culture of zygotes in vitro [10–12]. Zygotes that develop poorly in vitro (e.g., the C57BL/6 strain) show a marked up-regulation and nuclear accumulation of TRP53 in the resulting blastocysts, while this does not occur during development in vivo. Embryos that are null for TRP53 (Trp53−/−) show a marked increase in their developmental potential following culture from the zygote stage, showing that the increased TRP53 expression is responsible for a significant component of the loss of developmental potential of embryos subjected to culture in vitro . Embryos from hybrid mice (e.g., B6CBF1) are relatively resistant to the effects of culture, as assessed by their growth rate in vitro and their viability upon embryo transfer. The amount of TRP53 expressed in hybrid blastocysts is modest following being placed in culture. This differential expression of TRP53 by embryos provides a basis for the well-known strain-dependent differences in the susceptibility of embryos to culture.
Metabolic disturbances can also activate TRP53-mediated early embryopathy. Hyperglycemia, secondary to induced diabetes, causes an increased incidence of cell death in embryos with a consequent reduced rate of development. This phenotype is partially ameliorated by the deletion of the Trp53 gene in the mouse embryo [13, 14]. Inbred zygotes cultured to the blastocyst stage show an accumulation of TRP53 within the nuclei. TRP53 is a transcription factor, and its increased expression and nuclear localization results in a TRP53-dependent accumulation of BAX, indicating that it is transcriptionally active under these conditions . Hyperglycemia also results in increased BAX expression in embryos . A study of human embryos produced by intracytoplasmic sperm injection shows that TRP53 expression occurs at high levels within the nucleus of embryos that are degenerate or retarded in development, but is generally expressed at much lower levels in embryos of apparently normal morphology and growth rates .
Transcription of Trp53 is under the regulation of a range of transcription factors , including positive regulators, for example, transcriptional enhancer factor (TEF-4; officially known as TEA domain family member 2, TEAD2) and transacting transcription factor 1, and negative transcriptional regulators, for example, paired box protein-1, Y box protein 1, or Kruppel-like factor 4. A range of cell stressors, including genotoxic stress, can induce Trp53 transcription in somatic cells . In human preimplantation embryos produced by in vitro fertilization, a negative association between an embryo's Trp53 mRNA concentration and its morphology and rate of development is observed [16, 17]. Thus, embryos of the best morphological grading have the least Trp53 expression. This may indicate that the stressors of culture act via the induction of Trp53 gene expression.
In many settings, it has been shown that regulation of TRP53 expression occurs primarily posttranslationally [18–20] by the regulation of its half-life. TRP53 is subject to rapid ubiquitin-mediated degradation by the 26S proteosome. A range of stressors can suppress this rapid turnover of TRP53, allowing the TRP53 levels within a cell to rapidly increase and accumulate. Transformed mouse 3T3 cell double minute 2 (MDM2) functions as an ubiquitin ligase E3 toward itself and TRP53. It is an essential mediator of TRP53 ubiquitination and degradation . Absence of MDM2 (Mdm2−/−) in the preimplantation embryo results in their death, while a simultaneous lack of TRP53 (Mdm2−/−Trp53−/− compound mutant) rescues embryos from this lethality [22, 23]. This result infers an essential role for MDM2-mediated degradation of TRP53 in controlling preimplantation embryo survival under normal circumstances. MDM2 is commonly activated through its phosphorylation by RAC-alpha serine/threonine-protein kinase (AKT, also known as protein kinase B) that is in turn activated by binding to phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 is generated by the actions of phosphatidylinositol-3 kinase (PI3 kinase). Activation of PI3 kinase is commonly coupled to ligand-activated membrane receptors. It has not yet been determined whether this mechanism governs the level of TRP53 expression in the preimplantation embryo.
This study assesses the relative roles of Trp53 transcription and MDM2-mediated regulation of TRP53 expression in the embryo's response to the stresses experienced during culture in vitro. The study finds no evidence for increased transcription of Trp53 under culture conditions that lead to increased TRP53 expression. It did find that activation of MDM2 occurs via a trophic factor/PI3 kinase/AKT-dependent pathway, and this activation is perturbed in susceptible embryos during culture. The study shows that the maintenance of TRP53 latency in culture by the actions of a ligand-induced receptor-dependent PI3 kinase/AKT/MDM2 signaling pathway is one requirement for the normal autonomous development and survival of the preimplantation embryo.
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. Mice were inbred (C57BL/6J; B6); hybrid (C57BL/6J × CBA/He; B6CBF1); and Trp53−/− (B6.129S2-Trp53tm1Tyj strain, extensively backcrossed with B6 strain) Ptafr−/− mice were provided by Dr. S. Ishii, Department of Biochemistry and Molecular Biology, University of Tokyo. Females were superovulated with 5 IU eCG (Folligon, Intervet International, Boxmeer, The Netherlands) followed 48 h later by 5 IU hCG (Chorulon, Intervet). All were bred and maintained in the Gore Hill Research Laboratories (Royal North Shore Hospital, St Leonards, North South Wales) under 12L:12D cycle and had access to food and water ad libitum. Females were paired with males of proven fertility following hCG injection. Pregnancy was confirmed by the presence of a copulation plug the following morning (Day 0.5).
Embryos were flushed from the reproductive tract with Hepes-buffered modified human tubal fluid medium (Hepes-HTF) and cultured in modified HTF medium (mod-HTF) . All components of the media were tissue culture grade (Sigma, St. Louis, MO). Media contained 3 mg BSA/mL unless otherwise stated (CSL Ltd., Melbourne, Australia). Collection times post-hCG were as follows: oocytes or zygotes, 20–21 h; 2-cell embryos, 42 h; 8-cell embryos, 66 h; or blastocysts, 90 h. Zygotes were freed from their surrounding cumulus cells by brief exposure to 300 IU hyaluronidase (Sigma) in Hepes-HTF. Embryos were recovered in minimal volume and assigned to various treatments as required in mod-HTF. Embryos were cultured in 10 μl volumes in 60-well plates (LUX 5260, Nunc, Naperville, IL) overlaid by a depth of approximately 2 mm heavy paraffin oil (Sigma). Culture was at 37°C in 5% CO2 for the periods indicated in individual experiments. The developmental stage and morphology of embryos was assessed by visualizing the embryos with an inverted phase contrasted microscope (Nikon Diaphot, Nikon Corporation, Tokyo, Japan) at 24-h intervals after zygote collection.
The following pharmacologically active agents were used in indicated experiments: Paf (equal mixture of 1-o-octadecyl and hexadecyl-2-acetyl-sn-glycero-3-phosphocholine; Sigma); Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol 2-[(R)-2-O-methyl-3-O-octadecylcarbonate], Calbiochem, Alexandria, Australia); deguelin ((7aS,13aS)-13,13a-dihydro-9,10-dimethoxy-3,3-dimethyl-3H-bisbenzopyrano[3,4-b:6′,5′-e]pyran-7(7aH)-one; Sigma); LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, Calbiochem); Nutlin-3 (racemic mix of Nutlin-3a and Nutlin-3b, (+/−)-4-[4,5-bis-(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl]-piperazin-2-one; Calbiochem); and Pifithrin-α (2-(2-imino-4,5,6,7-tetrahydrobenzothiazol-3-yl)-1-p-tolylethanone; Calbiochem).
The blastocysts that were analyzed were washed three times in PBS, pH 7.4 (Sigma), transferred individually into 5 μl 1× PCR buffer II (final 1× concentrations: 50 mM KCl, 10 ml Tris-HCl, pH 8.3, Applied Biosystems) supplemented with 4 mg/ml glycogen (Roche) and 1 U/μl RNase inhibitor (Applied Biosystems) and immediately frozen in liquid nitrogen. The samples were lysed by thawing on ice and vortexed for 15 sec. Two more freeze, thaw, and vortex cycles were carried out.
Primer annealing to RNA occurred by the addition of 5 μl of a premixed 2× solution containing random hexamers (Roche), MgCl2, deoxyribonucleotide triphosphates (dNTPs; Applied Biosystems), and PCR buffer per 5 μl of sample such that the resulting solution was 1× PCR buffer II (Applied Biosystems, final 1× concentrations: 50 mM KCl, 10 mM Tris-HCl, pH 8.3), supplemented with 2 mg/ml glycogen (Roche), 0.5 mM each dNTP, 2.4 μM random hexamers, and 4.7 ml MgCl2. The RNA was denatured (65°C, 10 min), and placed on ice. The sample was supplemented with 1.25 U/μl MuLV Reverse Transcriptase (Applied Biosystems) and 0.5 U/μl RNase inhibitor (Applied Biosystems), and incubated at 25°C for 10 min for primer annealing and initial cDNA synthesis, and at 42°C for 40 min for completion of cDNA synthesis. The cDNA samples were stored at −20°C.
Real-time reverse transcriptase PCR analysis was performed in a Rotor Gene 3000 Real Time Thermal Cycler (Corbett Life Science, Sydney, Australia). Gene specific primers and internal Taqman probes were used: Trp53 forward primer 5′ CAG CGT GGT GGT ACC TTA TG 3′, reverse primer 5′ CCC CAT GCA GGA GCT ATT AC 3′, yielding a 91-bp product (lacking a 402-bp intron) with an internal probe of 5′ 6-6-carboxyfluorescein (FAM)-CTC AGA GCC GGC CTC GGG–5-carboxytetramethylrhodamine (TAMRA) 3′; Actb forward primer 5′ CTA AGG CCA ACC GTG AAA AG 3′, reverse primer 5′ GTA CGA CCA GAG GCA TAC AG 3′, yielding a 109 bp product (lacking 454 bp intron) with an internal probe of 5′ hexachlorofluorescein (HEX)-TGA AGG TCT CAA ACA TGA TCT GGG TCA–Black Hole Quencher 1 (BHQ)-1 3′ (all primers from Sigma-Genosys, Castle Hill, Australia).
PCR was performed in 25-μl reactions in PCR buffer II (Applied Biosystems), supplemented with cDNA template (3.3 μl per 25 μl reaction). The PCR cycling conditions were: 95°C for 10 min, then 40 cycles of: 95°C for 15 sec, 60°C for 30 sec, and 72°C for 45 sec; readings were acquired on channels appropriate for FAM and HEX labels.
A threshold was set where the amplification was close to the reaction's maximum rate and where negative controls were not significant. The threshold cycle (Ct) was used to calculate the relative quantity of the gene of interest in the cDNA sample. Samples where the Ct of either Trp53 or Actb was outside the known quantitative range (as indicated by the standard curves) were excluded from further analysis. The delta Ct (i.e., = Ct[Trp53] − Ct[Actb]) is a measure of relative changes in Trp53 mRNA content of the embryo. Data was normalized to the freshly isolated blastocysts Trp53 mRNA relative level by subtracting the delta Ct of each sample. The value of 2−(normalized delta Ct) is a measure of relative Trp53 mRNA expression, shown in Figure 1D.
Western blot analysis was performed as previously described . Embryos were collected and washed three times in cold PBS and transferred in a maximum volume of 1.5 μl PBS into 1.5 μl of 2× extraction buffer supplemented with protease and phosphatase inhibitors (2× PBS, 2% (v/v) Triton X-100, 24 mM deoxycholic acid, 0.2% (w/v) SDS, 20 mM NaF, 20 mM Na4P2O7, 2 mM PMSF, 3.08 μM aprotinin, 42 μM leupeptin, and 2.91 μM pepstatin A; all from Sigma). The embryos were lysed by three cycles of freezing in liquid nitrogen and thawing (with vortexing). Protein samples were diluted with 1 μl of 5× Laemmli buffer (50 mM Tris-HCl, 5 mM EDTA, pH 8.0, 12.5% (w/v) SDS, 0.05% (w/v) bromophenol blue, and 25% beta-mercaptoethanol) (Sigma), incubated 10 min at 60°C, and size separated using 20% homogenous SDS PAGE (GE Healthcare, Rydalmere, Australia) on a PhastSystem apparatus (GE Healthcare) or using Bio-Rad minigels (Bio-Rad Laboratories, Hercules, CA). Proteins were blotted into polyvinylidene fluoride (PVDF) membranes (Hybond-P, GE Healthcare) in a semidry blotting apparatus overnight using transfer buffer (12 mM Tris, pH 7.0, 96 mM glycine [Sigma], and 20% [v/v] methanol [Merck KGaA, Darmstadt, Germany]). Nonspecific binding was blocked by 5% (w/v) skim milk in PBS supplemented with 0.05% (v/v) Tween-20 (PBST) at room temperature for 1 h. Membranes were probed overnight at 4°C in 5% skim milk in PBST with primary antibody (1:400 rabbit anti-Ser-166 phosphorylated MDM2 (pMDM2) polyclonal IgG; Cell Signaling Technology, Beverly, MA) or with 1:1000 rabbit anti-beta-actin (Sigma). In experiments in which both pMDM2 and actin were assessed, the gel was cut into two pieces along a line corresponding to approximately 60 kDa. The lower molecular weight sections were exposed to the anti-pMDM2, and the higher molecular weight sections to anti-actin. Primary antibodies were detected with a horseradish peroxidase-conjugated secondary antibody applied for 1 h at room temperature. Anti-pMDM2 was developed with 1:2 dilution of Femto Maximum Chemiluminescent Substrates (Pierce, Rockford, IL) for 5 min at room temperature, and anti-actin was detected with 1:3 dilution with SuperSignal West Pico Substrate (Pierce). Membranes were exposed to CL-XPosure x-ray film (Pierce). Analysis was performed on groups of 30 embryos. The relative optical density of blots was measured by the Image-Pro Plus histogram program (Media Cybernetics Inc., Silver Spring, MD). Total density of each blot in three independent replicates was measured.
Embryos were washed three times in PBS with 0.1% (w/v) BSA, 0.1% (v/v) Tween-20, and 0.2% (w/v) sodium azide (washing buffer), fixed with freshly prepared 2% (v/v) paraformaldehyde (Sigma) in PBS (pH 7.4) for 30 min, and then permeabilized with 2% paraformaldehyde with 0.3% Tween-20 (Sigma) at room temperature for 30 min. Embryos were washed three times in washing solution and then were blocked in PBS containing 2% BSA and 30% serum for 3 h. They were stained overnight at 4°C with primary antibodies or an equivalent concentration of isotype control nonimmune immunoglobulin (negative control). Primary antibody was detected by incubation of embryos with secondary antibodies coupled to fluorescein isothiocyanate or Texas Red in PBS, 2% BSA for 1 h at room temperature. In some cases, the nuclei were counterstained with 0.1 μg/ml propidium iodide in PBS. Whole section immunolocalization was performed with mercury lamp UV illumination and epifluorescence on a Nikon Optiphot microscope with Olympus DPlan Apo 40X UV oil objective. Optical sectioning was performed with a Bio-Rad Radiance Confocal microscope, using a Nikon Plan Apo 60X/1.4 oil emersion objective, as previously described . Images were captured using Lasersharp 2000, Version 4.0 (BioRad, Sydney, Australia). Microscope and laser settings were adjusted such that no fluorescence was observed with nonimmune controls. All the test specimens were observed with these same settings. The primary antibodies and their concentrations were: 1:300 anti-TRP53 (Ab-7) polyclonal antibody (Oncogene Research Products, Calbiochem); 1:200 mouse anti-MDM2 (SMP14) monoclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA); and 1:400 rabbit anti-Ser-166 pMDM2 polyclonal IgG (Cell Signaling Technology). In all the experiments, negative control staining used equivalent concentrations of nonimmune IgG. For all individual experiments, microscope, data-capture, image analysis, and manipulation settings were identical for all treatments, including negative controls.
Statistical analyses were performed on SPSS statistical package (version 11.5, SPSS, Chicago, IL). The proportion of embryos developing to a given developmental landmark following culture in vitro was assessed by binary logistic regression analysis, treating the proportion developing to a given developmental landmark as the dichotomous dependent variable and the treatment and experimental replicate as covariates in the model. The effects of the factors, strain, and treatment on the relative concentration of RNA (dependent variable) was tested by univariate analysis. Difference in the optical density of Western blot analysis was tested by Student t-test.
Mouse embryos cultured from the zygote stage are generally recognized as being more susceptible to the adverse effects of culture than embryos cultured from the 2-cell stage. Inbred strain (B6) and hybrid embryos (B6CBF2) were cultured from either the zygote or 2-cell stage for 96 or 72 h, respectively. Embryos were cultured individually in 10 μl drops of media. B6 embryos cultured from the 2-cell stage had a significantly higher (P < 0.01) rate of development to the blastocyst stage than embryos cultured from the zygote stage for 96h (Fig. 1A). This differential was not observed for B6CBF2 embryos (P > 0.05) (Fig. 1A). B6 blastocysts cultured from the zygote stage showed a pattern of increased TRP53 staining compared with B6 embryos cultured from the 2-cell stage (Fig. 1B). This difference was not obvious for B6CBF2 embryos cultured from either developmental stage (Fig. 1B). The reduction in the rate of development of B6 embryos cultured from the zygote stage was partially reversed (P < 0.05) by the addition of a TRP53 inhibitor (Pifithrin-α)  to culture media (Fig. 1C). The inhibitor caused no change in the rate of development of hybrid embryo development compared with controls (P > 0.05) (Fig. 1C), a result consistent with the low level of TRP53 expresssed in these embryos. Thus, under conditions that caused a marked increase in the expression of TRP53 (culture of B6 zygotes), pharmacological inhibition of TRP53 improved the viability of embryos.
There was no effect of mouse strain on the amount of Trp53 mRNA expressed relative to Actb expression (P > 0.05), nor was there a difference in Trp53 expression between blastocysts that had been cultured from the zygote stage or 2-cell stage (P > 0.05) (Fig. 1D). Somewhat surprisingly, the relative levels of Trp53 expression in blastocysts collected directly from the reproductive tract was significantly higher for both strains (P < 0.001) than either of the corresponding cultured groups (Fig. 1D). Thus, conditions that resulted in increased levels of TRP53 expression were not accompanied by increased Trp53 RNA expression.
An important determinant of the level of TRP53 expression in cells is its rate of degradation, and this is largely determined by the activity of the protein ligase E3, MDM2. Phosphorylation of MDM2 (at Ser-166 or −188) can result in the activation of its protein ligase activity. Phosphorylated MDM2 was detected by Western blot analysis of embryos throughout the preimplantation phase of development in hybrid embryos and in both fresh and cultured B6 embryos (Fig. 2, inset). Immunolocalization showed that relative to the level of MDM2 staining, pMDM2 staining was lower in B6 blastocysts resulting from zygote culture than in either freshly collected hybrid or B6 blastocysts, or hybrid blastocysts resulting from culture of zygotes (Fig. 2, main). Thus, conditions that resulted in poor embryo viability (i.e., culture of B6 blastocyst) also resulted in a diminished level of MDM2 phosphorylation. It is noteworthy that where the expression of pMDM2 was low (cultured B6), the general pattern of staining was for expression throughout the cells of the embryo. By contrast, treatments that led to high levels of pMDM2 expression showed a general pattern of enhanced cytoplasmic staining in many cells.
AKT causes Ser-166/Ser-188 phosphorylation of MDM2 . Compared to untreated embryos, treatment of B6CBF2 zygotes with an inhibitor of PI3 kinase (LY294002) or AKT (Akt inhibitor) (Fig. 3A) caused an up-regulation of expression and nuclear localization of TRP53. An autocrine embryotrophin (Paf) induces the formation of PIP3 by activation of PI3 kinase resulting in the downstream activation of AKT [28, 29]. The absence of the receptor for Paf (Ptafr−/−) reduced the rate of development of zygotes in vitro  and induced the increased expression and nuclear localization of TRP53 in zygotes cultured for 96 h (Fig. 3A).
Paf is known to activate the PI3 kinase/AKT pathway during the zygote to 2-cell stage; thus, it was of interest to note that expression of pMDM2 was highest at the 2-cell stage (Fig. 2, inset). Zygotes were cultured to the 2-cell stage in the presence or absence of Paf, and the expression of pMDM2 was assessed. Western blot analysis showed that Paf induced an increase in the rate of MDM2 phosphorylation (Fig. 3B) without causing any change in the expression of actin. In a further analysis, we showed that inhibitors of PI3 kinase (LY294002) and AKT (deguelin) reduced the staining of pMDM2 induced by treatment of embryos with Paf (Fig. 3C), also without any consistent change in actin. These results indicate that pMDM2 staining in these embryos was at least partially under the regulation of the PI3 kinase and AKT pathway.
An inhibitor of MDM2 (Nutlin-3)  induced a dose-dependent decrease in the proportion of B6CBF2 hybrid zygotes developing to the blastocyst stage in vitro (P < 0.001) (Fig. 4A). Blocking MDM2 activity with Nutlin-3 also exacerbated the culture-induced embryopathy observed in B6 embryos, but this did not occur in B6 background embryos lacking the Trp53 gene (Trp53−/−) (Fig. 4B)..
The study shows that changes in the level of TRP53 expression in embryos that were highly susceptible to the adverse effects of culture occurred independently of an increase in the expression of Trp53 mRNA. Inhibition of the PI3 kinase/AKT/MDM2 pathway mediated by embryotrophic factors resulted in increased TRP53 expression and TRP53-dependent embryopathy. The study indicates that trophic stimulation of the early embryo by this pathway plays an important part in maintaining the latency of TRP53 expression. This latency is required for the normal development and survival of the preimplantation embryo.
This study shows that the level of TRP53 expression in the blastocyst is primarily regulated by the activity of MDM2, which in turn is regulated by the activity of PI3 kinase and AKT (summarized in Fig. 5). It is generally considered that TRP53 and MDM2 exist as an autoregulatory feedback loop; MDM2 transcription may be induced by TRP53, and MDM2 in turn binds to the N-terminal transactivation domain of TRP53, thereby inactivating TRP53 transcriptional activity [21, 31]. The net effect of the feedback loop is to maintain a low level of TRP53 expression and activity in normal cells. While this negative feedback mechanism is considered a major regulator of TRP53 expression in cells, the rate of transcription of Trp53 can also influence its activity. The current study shows that the level of TRP53 expressed in the mouse blastocyst is not primarily governed by changes in the level of its RNA. While embryo culture induced increased TRP53 expression, this was not associated with increased transcription of the Trp53 gene. Indeed, Trp53 expression was lower in embryos following their culture. This may be a manifestation of the broader perturbation of transcription that apparently occurs in the early embryo as a consequence of culture in vitro [32–35]. The relatively high levels of Trp53 expression in blastocysts collected from the reproductive tract was surprising. TRP53 is known to exert an autoregulatory positive feedback in some cell types . Hence, it might have been expected that transcription would be highest in cells expressing high TRP53 (culture B6). Yet many other transcription factors exert direct positive and negative regulatory actions on Trp53 expression, creating a complex regulatory network. The large effect of all cultures on Trp53 expression indicates that culture influences this regulatory network in an as yet unidentified manner.
Our results do not exclude the possibility that within individual embryos Trp53 is related to the morphology of that embryo, as has been observed in the human [16, 17], however, that question was not addressed in this study. The results do show that on a population basis, the level of Trp53 expression was not a primary determinant of embryo development or TRP53 expression.
The current study confirms the findings of Li et al.  that culture of B6 strain zygotes caused the latency of TRP53 expression to be breached and shows that pharmacological inhibition of TRP53 (Pifithrin-α) reduced the adverse effects of culture. The study also showed that increased TRP53 was associated with an increased expression of pMDM2. We show that pharmacological inhibition of MDM2 induced early embryopathy in hybrid embryos, which were normally resistant to the stressors of culture. In B6 background embryos, the reduced embryo development induced by blocking MDM2 with Nutlin-3 did not occur in embryos that lacked the Trp53 gene (Trp53−/− embryos). The essentially complete absence of an effect of Nutlin-3 in Trp53−/− embryos indicates that TRP53 is the major downstream target of MDM2 in this experimental setting. This result indicates that the early lethality of Mdm2−/− embryos (and the reversal of this effect in Mdm2−/− Trp53−/− compound mutant embryos) [22, 23] was a consequence of the regulation of TRP53 expression by MDM2.
The greater sensitivity of the zygote of some strains to the adverse effects of culture has been previously reported and found to be at least partially due to the deprivation of autocrine trophic ligands that occurs when embryos are cultured from this stage of development [1, 2]. The early embryo responds to a range of trophic ligands, including Paf [36–38]. Blockade of trophic signaling reduces normal embryo development, and in this study we show that selective blockade of Paf signaling (Ptafr−/− embryos) results in their increased TRP53 expression. It is known that the culture of zygotes in vitro reduces the amount of autocrine stimulation the embryo receives (and in vitro the embryo is not exposed to the range of paracrine mediators released into the reproductive tract) (for review, see ), and this deprivation is exacerbated when embryos are cultured individually as they were in this study. This trophic deprivation is thought to make a significant contribution to the loss of viability that occurs upon the culture of zygotes and also serves as a good model for studying the actions of autocrine trophic ligands. It is noteworthy that mouse embryos cultured from the 2-cell stage are not as sensitive to reduced autocrine stimulation [1, 2], and in this study we show this is associated with the relative maintenance of TRP53 latency.
Several key proofs provided in this report rely of the use of pharmacological inhibitors. This strategy must always be interpreted with caution given the possibility of nonselective actions. The selectively of PI3 kinase inhibitors at the doses used in this study has been established and reviewed , and is consistent with the early embryopathy observed in Pik3cb−/− embryos. The embryopathy induced by Nutlin-3 is in the dose range found to be selective for this drug  and is consistent with the known embryopathy of Mdm2−/− embryos [22, 23]. The partial rescue of the viability of B6 zygotes following culture by Pifithrin-α is consistent with a beneficial effect of the Trp53−/− genotype following the culture of zygotes . We have used two structurally unrelated inhibitors of AKT to reduce the risk of off-target actions. Akt-inhibitor is a phosphatidylinositol ether analog that potently and selectively inhibits AKT (IC50 of 5 μM), and it acts only as a week inhibitor on PI3 kinase (IC50 of 83 μM). Deguelin by contrast is a naturally occurring rotenoid of the flavonoid family. It is structurally and functionally different from Akt-inhibitor and has an IC50 of 10 nM. It has previously been demonstrated that these two antagonists causes reduced zygote development in these selective dose ranges .
Autocrine signals induce receptor-mediated activation of PI3 kinase [28, 29, 41]. PI3 kinase induces the phosphorylation of membrane inositol phospholipids, resulting in the formation of D-3′ phosphoinositides. The most important reaction may be the conversion of phosphatidylinositol (4,5) bisphosphate to phosphatidylinositol (3,4,5)-trisphosphate . PIP3 acts as a docking site for a range of proteins containing the pleckstrin homology (PH) domain . The generation of PIP3 recruits proteins containing PH domains to the membrane, facilitating their activation. AKT is activated as a consequence of PI3 kinase activity. The resulting PIP3 serves as a docking site for AKT where it is phosphorylated by 3-phosphoinositide-dependent kinase 1 .
The preimplantation embryo expresses multiple isoforms of PI3 kinase  and AKT . Inhibitors of either PI3 kinase or AKT prevent normal preimplantation embryo development in vitro [28, 45, 46], leading to increased rates of cell death and a reduced number of cells populating the embryo. The embryopathy induced by the inhibition of PI3 kinase or AKT can be partially reversed by the addition of exogenous Paf to embryo culture media [28, 44]. Paf induces the activation of PI3 kinase, resulting in the generation of PIP3 and causing the phosphorylation of AKT (Ser-473 phosphoAKT) in the early embryo . In this study, we show that the phosphorylation of MDM2 is regulated in part by the actions of Paf, PI3 kinase, and AKT. Inhibition of each component of the pathway reduces pMDM2 and increases TRP53, and consequent embryopathy. A range of putative embryotrophins activate PI3 kinase in preimplantation embryos. Both insulin  and transforming growth factor alpha  are reported to act in a PI3 kinase-dependent fashion in the preimplantation embryo. Injection of mRNA coding for a constitutively active myristoylated AKT into mouse zygotes enhances their rate of cell-cycle progression, and conversely, mRNA of kinase-deficient AKT delays the entry of embryos into mitosis. It has also been found that AKT induces the phosphorylation of M-phase inducer phosphatase 2, and hence induces maturation promoting factor activity . Thus, evidence from a range of sources support the presence and actions of the PI3 kinase/AKT pathway during the preimplantation phase of development. It is likely that the sequential and overlapping actions of a range of ligands acting via this pathway throughout the preimplantation stage cooperate. This study shows that maintenance of the latency of TRP53 expression via the action of MDM2 is one target of action of this pathway.
AKT activation results in its translocation to the nucleus , allowing MDM2 phosphorylation to occur in the nuclear compartment . MDM2 is a phosphoprotein, and its phosphorylation state determines its concentration and activity. Phosphorylation of MDM2 can protect it from self-ubiquitination, thereby stabilizing the protein, allowing it to accumulate within cells. MDM2 posses a nuclear export sequence that results in the export of the protein from the nucleus . This export function is required for the export and degradation of TRP53, although the two export events may be independent . The interactions between MDM2 and TRP53 are complex, and different modes of regulation may occur, depending upon the stoichiometric relationship between these two autoregulatory partners . In this study, we found that much of the pMDM2 detected in fresh blastocysts was cytoplasmic, indicative of active nuclear export, while the lower expression in cultured B6 blastocysts was not as completely exported from the nucleus, a result consistent with elevated TRP53 expression.
MDM2 is a target for several kinases. Phosphorylation of MDM2 by AKT results in its activation, yet phosphorylation of MDM2 at other sites by a range of other kinases involved in genotoxic stress responses (e.g., by DNA-dependent protein kinase at Ser-17 , cyclin A-dependent kinase at Thr-216 , casein kinase II at Ser-267 , and ataxia telangiectasia mutated protein kinase at Ser-395 ) result in the inhibition of MDM2. Furthermore, cyclin-dependent kinase inhibitor 2A acts to sequester MDM2 to the nucleolus, thus diminishing its interaction with TRP53. MDM2 integrates information from many sources to govern the final concentration and activity of TRP53 in the cell. Further studies are required to investigate the role of alternative pathways in the regulation of TRP53 expression in the early embryo.
This study confirms the role of TRP53 in the stress-induced embryopathy that occurs in some embryos following culture. It shows that a mechanism for the control of TRP53 expression occurs via the negative-feedback loop between MDM2 and TRP53, and that a regulator of the feedback loop is the ligand-mediated activation of MDM2 via a PI3 kinase/AKT signaling pathway. Perturbation of this pathway allowed TRP53 to accumulate in embryos and resulted in their loss of viability. The latency of TRP53 expression is required for the normal development of the preimplantation embryo, and the actions of a survival signaling pathway are shown to be a mechanism for achieving this. The results of this study will help to develop an understanding of the causes of loss of embryo viability in mammalian species following the exposure of embryos to a range of exogenous stressors, including those imposed during forms of assisted reproductive technologies.
We thank Dr. R. Christensen for maintaining animal pedigrees, the staff of the Gore Hill Research Laboratories for the breeding and care of animals, Greg Mulhearn for assistance in preparation of figures, and N. Gunay and A. Cahana for undertaking preliminary experiments not reported here. I also thank Professor G. Lozano, MD Anderson Cancer Centre, for assistance in performing some preliminary analysis of this question.
1Supported by grants from the Australian Health and Medical Research Council. H.D.M. was supported by a NHMRC C J Martin Fellowship, and V.C. was supported by an Australian Postgraduate Award.