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BOR - Papers in Press, published online ahead of print August 3, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.042754
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BIOLOGY OF REPRODUCTION 73, 1094–1101 (2005)
DOI: 10.1095/biolreprod.105.042754
© 2005 by the Society for the Study of Reproduction, Inc.

Research Article

Survivin Contributes to the Anti-Apoptotic Activities of Transforming Growth Factor alpha in Mouse Blastocysts Through Phosphatidylinositol 3'-Kinase Pathway

Kazuhiro Kawamura 1 2, Jun Fukuda 2, Yasushi Shimizu 2, Hideya Kodama 3, and Toshinobu Tanaka 2

Department of Obstetrics and Gynecology,2 Akita University School of Medicine, and Department of Maternity and Childnursing,3 Akita University School of Health Science, Akita, 010-8543 Japan

ABSTRACT

Transforming growth factor alpha (TGFA) is produced by epithelial cells in the oviducts and uteri and has the potential to act as an anti-apoptotic factor on preimplantation embryos expressing its receptor. Previously, we demonstrated that survivin (also known as BIRC5), an anti-apoptotic gene expressed in mouse preimplantation embryos, protects embryos from apoptosis. In this study, we investigated the role of survivin on TGFA-mediated inhibition of apoptosis in mouse blastocysts. Under the suboptimal conditions produced by single embryo culture, blastocysts showed an increase of apoptosis that correlated with a decrease of survivin expression. TGFA treatment significantly decreased apoptosis and increased the levels of survivin mRNA in a dose-dependent manner in blastocyst, and conversely, these activities were neutralized by an anti-TGFA antibody. Antibody treatment alone exerted little effect on either the occurrence of apoptosis or the levels of survivin mRNA. Upregulation of survivin expression by TGFA treatment was insignificant before the blastocyst stage. Using an antisense approach, we examined whether upregulation of survivin is responsible for the anti-apoptotic effect of TGFA in blastocysts. Apoptosis was inhibited by TGFA treatment in blastocysts, but the effect was abrogated by cotreatment with antisense oligonucleotides directed against survivin. These data suggest that survivin contributes to the anti-apoptotic activities of TGFA in blastocysts. We also found that the upregulation of survivin expression was mediated by activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Thus, TGFA inhibits apoptosis in mouse blastocysts through upregulation of survivin expression via the PI3K pathway.

apoptosis, early development, embryo, female reproductive tract, growth factors

INTRODUCTION

Transforming growth factor alpha (TGFA, also known as TGF{alpha}) is produced by epithelial cells in the oviducts and uteri of diverse species, including mice and humans [1–7]. In the mouse, the expression levels of TGFA and its precursor increased in the luminal epithelium of the receptive uterus before implantation [3, 8]. TGFA is structurally related to epidermal growth factor (EGF) and competes for binding to the EGF receptor (EGFR) [9]. Activation of the EGFR involves its dimerization, conformational change, and stimulation of the tyrosine kinase activity of the intracellular domain [10]. Fertilized mouse and human oocytes and preimplantation embryos express the TGFA ligand and EGFR, and these two signaling pathway components continue to be expressed in all subsequent stages of development [11, 12]. In blastocyst stage embryos, TGFA is produced by the inner cell mass (ICM) and the polar trophectoderm. EGFR is preferentially localized in the ICM and at the basolateral surface of the trophectoderm [13, 14]. Therefore, TGFA has the potential to affect the development of preimplantation embryos through the autocrine and/or paracrine pathways.

Apoptosis (or programmed cell death) is an essential physiologic process that occurs in almost all tissues [15, 16]. In preimplantation embryos, apoptosis is considered to be an important mechanism for removing unwanted cells, thereby maintaining embryo health or developmental potential [17]. Furthermore, the increase of apoptosis in preimplantation embryos associates with the degeneration of embryos during in vitro cultures [17]. In the in vitro culture environment, apoptosis of early embryos can be attributed to the lack of maternal factors, such as essential growth factors and cytokines released by maternal cells [17]. Although preimplantation embryos have a potential to develop up to the blastocyst stage in vitro, their development is slower, and postimplantation viability is lower than it is for embryos that develop in vivo [17, 18]. Furthermore, blastocysts developed in vitro often have increased number of apoptotic cells, and the phenomenon is more pronounced when two-cell-stage embryos are cultured to the blastocyst stage singly. The number of apoptotic cells in blastocysts cultured singly is 2-fold higher than in blastocysts cultured in groups [19]. Thus, it seems that for single embryo cultures, survival factors secreted by the preimplantation embryos themselves are diluted into an insufficient dose for inhibition of apoptosis.

The incidence of apoptosis in blastocysts appears to correlate with embryo quality [20]. The rate of resorption of fetuses is significantly increased in blastocysts with 2-fold higher apoptotic cells than for those in normally developed blastocysts [21]. Supplementation of the culture medium with several growth factors, such as TGFA, insulin-like growth factor (IGF)-I, and granulocyte-macrophage colony stimulating factor, significantly reduces apoptosis in mammalian blastocysts [17, 22]. Of these growth factors, TGFA has been shown to inhibit apoptosis induced by single embryo culture [19]. Furthermore, apoptosis is increased in TGFA-deficient mouse blastocysts [23]. These findings indicate that TGFA plays a key role in regulating apoptosis during preimplantation embryo development. However, the molecular mechanisms underlying the regulation of apoptosis in blastocysts by TGFA are unknown.

Previously, we demonstrated that survivin (also known as BIRC5), a member of the inhibitor of the apoptosis protein family, is an essential anti-apoptotic gene expressed in preimplantation embryos [24]. Survivin mRNA and protein were expressed in oocytes and throughout the development of preimplantation embryos. Embryos treated with antisense survivin oligonucleotides stopped developing at the morula or early blastocyst stages. At the cellular level, disruption of tubulin formation and the presence of abnormal nuclei, both of which are associated with apoptosis, were observed. In contrast, apoptosis induced by the antisense treatment is inhibited by caspase-3 and –9 inhibitors. These results indicate that survivin is expressed throughout early embryonic development and that it prevents apoptosis of these cells by inhibiting an apoptotic pathway involving the caspases.

The aim of this study was to investigate the role of survivin on TGFA-mediated inhibition of apoptosis in mouse blastocysts. We sought to determine whether upregulation of survivin is responsible for the anti-apoptotic effect of TGFA in blastocysts and to dissect the molecular pathway of TGFA-mediated inhibition of apoptosis in blastocysts.

MATERIALS AND METHODS

Collection and Culture of Mouse Preimplantation Embryos

Female IVCS mice, 9 wk old (Institute for Animal Reproduction), were superovulated with an intraperitoneal injection of 10 IU of equine chorionic gonadotropin (Sigma), followed 48 h later by 10 IU of human chorionic gonadotropin (hCG; Sigma). Two-cell-stage embryos were obtained by flushing the oviducts of the mated mice at 46–47 h after hCG injection. The embryos were washed three times with M2 medium (Sigma). Groups of 30 or single embryos were placed in 30-µl drops of M16 medium (Sigma) and covered by mineral oil with or without TGFA (Oncogene Science, Inc.). The embryos were then transferred to freshly prepared media every 24 h. Embryos were cultured 72 h up to the expanded blastocyst stage at 37°C in 5% CO2 in air.

To examine the specific effects of TGFA, single two-cell-stage embryos were cultured in M16 medium containing 0.2 µg/ml of neutralizing monoclonal mouse anti-TGFA antibody (Oncogene Science) with or without 0.1 pM of TGFA. The antibody and TGFA were incubated for 1 h at 37°C before addition of the medium. For controls, embryos were cultured in M16 medium alone, M16 medium with an equivalent concentration of nonimmune mouse IgG1 (Zymed, Inc.), or mouse IgG1 with 0.1 pM of TGFA.

To investigate the role of PI3K and nuclear factor kappa B (NF{kappa}B) as downstream mediators of TGFA-stimulated upregulation of survivin, single morulae were cultured for 24 h with or without 0.1 pM of TGFA in the absence or presence of a PI3K inhibitor, either 5 µM of LY294002 (Sigma) or 50 nM of wortmannin (Sigma), and in the absence or presence of an NF{kappa}B inhibitor, either 1 µM of pyrrolidine dithiocarbamate (PDTC; Wako Pure Chemicals, Inc.) or 1 mM N-acetyl-L-cysteine (NAC; Wako Pure Chemicals). The concentrations of the PI3K and NF{kappa}B inhibitors were based on previous studies of germ cells and early embryos [25, 26].

All procedures involving the care and use of animals were approved by the Animal Research Committee, Akita University School of Medicine.

Conventional RT-PCR

Methods for conventional RT-PCR of oocytes and preimplantation embryos have been described previously [24, 27, 28]. The primer for PI3K p110 was based on GenBank accession number NM_008839: forward 5'-CCATGGAACTCCTGGACTGT-3', reverse 5'-CACGGCAGTAGGACTCCAAT-3'. The primer for ß-actin has been described previously [24]. PCR reactions comprised 40 cycles of amplification, and cycling conditions of PI3K and ß-actin were as follows: denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, and elongation at 72°C for 30 sec. For positive controls, mouse placenta cDNA was amplified simultaneously. For negative controls, we included a sample in which water was substituted for mRNA.

Quantitative Real-Time RT-PCR

Quantitative real-time RT-PCR was performed to measure levels of survivin in mouse preimplantation embryos using a Light Cycler (Roche Diagnostics, Inc.) in accordance with the instructions of the manufacturer. Methods for mRNA extraction and reverse transcription for preimplantation embryos have been described previously [24, 27, 28]. Thirty embryos were used in the initial poly (A)+ mRNA isolation step. Primers and hybridization probes for survivin and glyceraldehyde-3-phosphate dehydrogenase (GAPD) were based on GenBank accession number AF115517 and NM_008084, respectively: survivin primers: forward 5'-AGCATAGAAAGCACTCCCCT-3', reverse 5'-CAATTGACTGACGGGTAGTC-3'; GAPD primers: forward 5'-TGAACGGGAAGCTCACTGG-3', reverse 5'-TCCACCACCCTGTTGCTGTA-3'; survivin probes: donor 5'-TGGACAGACAGAGAGCCAAGAACAAAAT-3', acceptor 5'-GCAAAGGAGACCAACAACAAGCAAAA-3'; GAPD probes: donor 5'-GCATCTTGGGCTACACTGAGGACC-3', acceptor 5'-GGTTGTCTCCTGCGACTTCAACAG-3'. Primers for survivin were designed to amplify only the full length of mouse survivin because it has been reported that the mouse survivin gene was transcribed into three alternatively spliced variants [29]. Fluorescent probes were designed to hybridize to target sequence in a head-to-tail arrangement on the same strand of amplified products. Donor probes were labeled at the 3' end using fluorescein, while acceptor probes were labeled at the 5' end with LCRed640 and modified at the 3' end by phosphorylation to block extension. The gap between the 3' end of the donor probe and the 5' end of the acceptor probe was one base. All primers and probes were synthesized and purified using reverse-phase high-performance liquid chromatography (Nihon Gene Research Laboratories). Real-time PCR products were validated by gel-isolation and cycle sequencing.

Quantitative real-time RT-PCR was performed in a total reaction volume of 20 µl/capillary for the LightCycler format. The 20-µl reaction mixture was composed of 1x FastStart Master Hybridization Probes mix (Roche Diagnostics); 4.0 and 3.0 mM of MgCl2; 0.3 µM and 0.5 µM of primer pairs for survivin and GAPD, respectively; 0.2 µM of fluorescein probe; 0.4 µM of LCRed 640 probe; and suitable dilutions of template cDNA. In all reactions, FastStart Taq DNA polymerase was activated by an initial denaturation at 95°C for 10 min followed by 40 cycles of amplification. Cycling conditions of survivin and GAPD were as follows: denaturation at 95°C for 10 sec; annealing at 60°C for 15 and 10 sec, respectively; and elongation at 72°C for 7 and 10 sec, respectively. Temperature transition rates of 20°C/sec were used for cycling. Real-time PCR monitoring was achieved by measuring the fluorescent signal at the end of the annealing phase for each cycle.

To determine the absolute copy number of target transcripts, cloned plasmid cDNAs for survivin and GAPD were used to generate a calibration curve. Briefly, total RNA was isolated from mouse placenta using the RNeasy Mini kit (Qiagen KK) according to the manufacturer's protocol. Reverse transcription was performed as described previously [24, 27, 28]. Target transcripts were amplified using conventional RT-PCR with specific primers. PCR cycling conditions were as follows: denaturation at 95°C for 3 min, followed by 30 cycles of denaturation at 95°C for 30 sec; annealing at 60°C for 30 sec; and extension at 72°C for 30 sec. PCR products were validated as described previously. Purified plasmid cDNA templates were measured, and copy numbers were calculated from absorbance at 260 nm.

A calibration curve was created by plotting the threshold cycle against the known copy number for each plasmid template diluted in log steps from 105 to 101 copies. Each run included standards of diluted plasmids to generate a calibration curve, a negative control without a template, and samples with unknown mRNA concentrations. Copy numbers for all unknown samples were determined by LightCycler software 3.1 (Roche Diagnostics). To correct for differences in both RNA quality and quantity between samples, data were normalized by dividing the copy number of the target cDNA by the copy number for GAPD [30].

Caspase-3 Assay for Detection of Apoptosis

To investigate apoptosis in cultured preimplantation embryos, activated caspase-3 was detected using the PhiPhilux G1D2 kit (OncoImmunin Inc.) as described previously [24]. In each experiment, embryos treated with 50 µM of staurosporine for 24 h before being incubated with caspase-3 substrate and embryos incubated without caspase-3 substrate were included as positive and negative controls, respectively. Based on the mean number of caspase-3-positive cells in blastocysts recovered from the uteri of pregnant mice at Day 4 after mating, 1.68 ± 0.34 (mean ± SEM), the blastocysts with more than three caspase-3-positive cells were defined as caspase-3-positive embryos.

Terminal Deoxynucleotidyl Transferase-Mediated dUDP Nick End-Labeling (TUNEL) Assay for Detection of Apoptosis

TUNEL assay was performed as described previously with slight modifications [19]. Briefly, zona pellucida-intact embryos were washed, fixed in 4% paraformaldehyde for 30 min at room temperature, and permeabilized in 0.5% Triton X-100 (Sigma) for 1 h at room temperature. Embryos were incubated with TUNEL reagent (Roche Molecular Biochemicals) for 1 h at 37°C in the dark. For positive controls, embryos were treated with 50 µg/ml Ribonuclease A (Fermentas, Inc.) for 20 min at 37°C before the TUNEL reaction. For negative controls, embryos were incubated with TUNEL reagent in the absence of the enzyme terminal deoxynucleotidyl transferease. Counterstaining was performed by incubating embryos with 100 µg/ml propidium and 50 µg/ml RNase A (Ambion, Inc.) for 20 min at room temperature. Embryos were washed and mounted with Slowfade light antifade solution (Invitrogen, Inc.). The fluorescence signals in embryos were visualized using a confocal laser scanning microscope (LSM 410; Carl Zeiss).

Embryo Culture with Survivin Antisense Oligonucleotides

Antisense oligonucleotides and controls directed to mouse survivin were designed and validated previously [24]. Single two-cell-stage embryos were cultured for 72 h up to the expanded blastocyst stage in M16 medium in the presence of either an antisense oligonucleotide at 50 µM or a missense oligonucleotide at 50 µM with or without 0.1 pM of TGFA. Control group of embryos were cultured in the absence of both antisense and missense oligonucleotides with or without 0.1 pM of TGFA.

Statistical Analysis

The one-way ANOVA coupled with Fisher's protected least significant difference test was used to evaluate differences in the proportion of caspase-3-positive embryos, the percentage of TUNEL-stained nuclei, and the relative levels of survivin mRNA in blastocysts. The Mann-Whitney U-test was performed for the comparison of the relative levels of survivin mRNA in each developmental stage of preimplantation embryos. Results are presented as means ± SEM of at least three separate experiments.

RESULTS

Suppression of Apoptosis in Blastocysts by TGFA

To examine whether TGFA can suppress apoptosis in mouse preimplantation embryos, groups of more than 30 or single two-cell-stage embryos were cultured in M16 medium alone, and single two-cell-stage embryos were cultured in the presence of increasing concentrations of TGFA. The embryos were allowed to develop to the expanded blastocyst stage and were then assayed for apoptosis as judged by caspase-3 activation, which is the last step of the apoptotic cascade and the one that leads to the cleavage of cellular substrates important for cell survival [31]. In expanded blastocysts, the proportions of caspase-3-positive embryos decreased when embryos are cultured in a group as compared with embryos cultured singly (Fig. 1a; P < 0.005). Under single embryo culture, embryos treated with TGFA showed a significant decrease in the proportion of caspase-3-positive embryos as compared with the group without TGFA treatment in a dose-dependent manner (Fig. 1a). The effect was most potent at 0.1 pM (P < 0.005) and decreased with a high dose (1.0 pM). In positive control embryos, almost all the cells showed activated caspase-3 signals, whereas no caspase-3-positive cells was observed in negative control embryos (data not shown). Furthermore, we performed TUNEL assay to confirm the effect of TGFA on apoptosis in blastocysts (Fig. 1b). Treatment with TGFA decreased the percentage of TUNEL-stained nuclei in blastocysts in a dose-dependent manner with similar tendency of the results of caspase-3 assay.


Figure 1
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  		FIG. 1. The effect of TGFA on apoptosis in mouse blastocysts. Apoptosis was assayed by detecting caspase-3 activity (a and c) or TUNEL staining (b) in three independent experiments in which 48–152 embryos were used for each treatment group. a and b) Dose-dependent effect of TGFA. Groups of more than 30 (C[>30]) or single two-cell-stage embryos (C) were cultured for 72 h in M16 medium alone, and single two-cell-stage embryos were cultured in M16 medium with increasing concentrations of TGFA. c) Specificity of the effect of TGFA on blastocysts. Single two-cell-stage embryos were cultured for 72 h in M16 medium alone (C), in M16 medium with 0.1 pM of TGFA in the absence (TGFA) or presence (TGFA+Ab) of neutralizing anti-TGFA antibody or nonimmune IgG1 (TGFA+IgG), or in M16 medium with only the anti-TGFA antibody (Ab) or nonimmune IgG1 (IgG). Data are expressed as the mean ± SEM. *P < 0.05; **P < 0.005 vs. C

Specificity of the suppression of apoptosis induced by TGFA was confirmed by introduction of the neutralizing monoclonal mouse anti-TGFA antibody (Fig. 1c). The antibody significantly inhibited the effect of TGFA (P < 0.005), and antibody treatment alone had no effect on the proportion of caspase-3-positive embryos in comparison to the group without TGFA treatment. For controls, treatment with nonimmune mouse IgG1 did not show any effects on the occurrence of apoptosis in blastocysts (Fig. 1c).

Effect of TGFA on Expression of Survivin in Blastocysts

Real-time quantitative RT-PCR was performed to determine the levels of survivin mRNA in mouse preimplantation embryos with or without TGFA treatment. In expanded blastocysts, the level of survivin mRNA was increased when embryos were cultured in a group as compared with embryos cultured singly (Fig. 2a; P < 0.005). Treatment with TGFA increased expression of survivin mRNA in a dose-dependent manner (Fig. 2a). The maximal effect was achieved at 0.1 pM (P < 0.005), whereas the effect decreased at a high dose (1.0 pM). Although no significant differences were identified, TGFA treatment tended to increase the level of survivin mRNA from morula stage embryos (Fig. 2b). Although baseline levels of survivin expression appear to increase with developmental stage of embryos, comparisons of survivin expression among developmental stages are not accurate because the amount of GAPD mRNA used as an endogenous standard to normalize differences in both RNA quality and quantity among samples was not constant throughout the preimplantation period [32].


Figure 2
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  		FIG. 2. Quantification of survivin mRNA levels in mouse blastocysts treated with TGFA by real-time RT-PCR. a) Dose-dependent effect of TGFA. Groups of more than 30 (C[>30]) or single two-cell-stage embryos (C) were cultured for 72 h in M16 medium alone, and single two-cell-stage embryos were cultured in M16 medium with increasing concentrations of TGFA. b) Time-course effect of TGFA. Single two-cell-stage embryos were cultured with or without 0.l pM of TGFA for 3 h (four-cell), 20 h (eight-cell), 43 h (morula), and 72 h (blastocyst). c) Specificity of the effect of TGFA on blastocysts. Single two-cell-stage embryos were cultured for 72 h in M16 medium alone (C), in M16 medium with 0.1 pM of TGFA in the absence (TGFA) or presence (TGFA+Ab) of neutralizing anti-TGFA antibody or nonimmune IgG1 (TGFA+IgG), or in M16 medium with only the anti-TGFA antibody (Ab) or nonimmune IgG1 (IgG). Data are expressed as the mean ± SEM. *P < 0.05; **P < 0.005

Specificity of the effects of TGFA on the upregulation of survivin mRNA was confirmed using neutralizing monoclonal mouse anti-TGFA antibody (Fig. 2c). The antibody significantly inhibited the TGFA-induced upregulation of survivin (P < 0.005), and the inhibitory effect was not observed in a control group in which nonimmune mouse IgG1 was substituted for anti-TGFA antibody. The antibody treatment alone exerted no significant effect on levels of survivin mRNA in comparison to controls.

Effects of TGFA on Apoptosis in Blastocysts Treated with Survivin Antisense Oligonucleotides

To examine whether the upregulation of survivin is responsible for the anti-apoptotic effect of TGFA in blastocysts, survivin was knocked down by antisense oligonucleotides. Under single embryo culture, treatment with TGFA significantly decreased the proportions of caspase-3-positive embryos (Fig. 3a) and TUNEL-stained nuclei of blastocysts (Fig. 3b) in untreated or missense-treated groups (P < 0.005). As expected from our previous studies [24], the group treated with antisense oligonucleotides showed a significant increase in the proportions of caspase-3-positive embryos (Fig. 3a) and TUNEL-stained nuclei of blastocysts (Fig. 3b) as compared with the group treated with missense oligonucleotides or the group without treatment (P < 0.0001). However, the inhibition of apoptosis by TGFA treatment was absent in survivin antisense-treated blastocysts (Fig. 3, a and b). Thus, these data suggest that survivin has an important role for the anti-apoptotic action of TGFA in blastocysts.


Figure 3
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  		FIG. 3. The effect of TGFA on apoptosis in survivin antisense targeted blastocysts. Apoptosis was assayed by caspase-3 activity (a) or TUNEL staining (b) in three independent experiments in which 45–88 embryos were used for each treatment group. Single two-cell-stage embryos were cultured in the presence of antisense oligonucleotides at 50 µM (antisense) or missense oligonucleotides at 50 µM (missense) with or without 0.1 pM of TGFA for 72 h. Control group of embryos (C) was cultured in the absence of oligonucleotides. Data are expressed as the mean ± SEM. **P < 0.005; {dagger}P < 0.0001

Temporal Expression of PI3K p110 mRNA in Mouse Oocytes and Preimplantation Embryos

RT-PCR was performed to detect mRNA for PI3K p110, the 110-kDa catalytic subunit of PI3K [33], in mouse oocytes and early embryos at different stages (two-cell, four-cell, eight-cell, morula, blastocyst, and hatched blastocyst). PI3K p110 mRNA was detectable as a 288-bp band in oocytes and at all the embryonic stages examined (Fig. 4). As loading controls, no significant differences were observed in the intensities of ß-actin amplification products among oocytes and preimplantation embryos at different stages.


Figure 4
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  		FIG. 4. RT-PCR detection of phosphatidylinositol-3-kinase (PI3K) p110 mRNA from mouse oocytes and each developmental stage of preimplantation embryos. For internal control, ß-actin was amplified simultaneously in each PCR reaction. Marker, {phi}x 174-Hae III digest; positive control, mouse placenta cDNA; negative control, without template cDNA

Blockade of TGFA-Induced Upregulation of Survivin by PI3K Inhibitors, Not NF{kappa}B Inhibitors

Because TGFA treatment did not significantly increase levels of survivin mRNA in preimplantation embryos before the morula stage, single morulae were cultured for 24 h to examine the roles of PI3K and NF{kappa}B as downstream mediators of TGFA-stimulated upregulation of survivin. When TGFA treatment was initiated in morula stage embryos, upregulation of survivin mRNA was similar to that from embryos in which treatment was started at the two-cell stage (Fig. 5, a and b). The ability of TGFA to upregulate survivin mRNA in mouse blastocysts was almost completely suppressed by cotreatment with either of the two PI3K inhibitors (LY294002 and wortmannin) with similar efficiency (Fig. 5a). In contrast, neither of the two NF{kappa}B inhibitors (PDTC and NAC) showed significant suppression of survivin expression in blastocysts (Fig. 5b). In the absence of TGFA treatment, the levels of survivin mRNA in blastocysts treated with either PI3K or NF{kappa}B inhibitors were comparable to those obtained in control cultures without inhibitor, confirming specificity of the inhibitor response to only those blastocysts receiving external stimulus coupled to PI3K activation (Fig. 5, a and b).


Figure 5
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  		FIG. 5. Suppression of TGFA induced upregulation of survivin by PI3K (a) and nuclear factor {kappa}B (NF{kappa}B) inhibitors (b) in blastocysts. Expression levels were determined by real-time quantitative RT-PCR. Single morula stage embryos were cultured for 24 h with or without TGFA and in the absence or presence of an inhibitor of PI3K, either 5 µM of LY294002 (LY) or 50 nM of wortmannin (WO), or an inhibitor of NF{kappa}B, either 1 µM of pyrrolidine dithiocarbamate (PDTC) or 1 mM N-acetyl-L-cysteine (NAC). Data are expressed as the mean ± SEM. **P < 0.005

DISCUSSION

In agreement with previous studies [19, 23], treatment with TGFA decreases the proportions of caspase-3-positive embryos and TUNEL-stained nuclei in blastocysts cultured singly, suggesting an important autocrine/paracrine role of TGFA for the inhibition apoptosis in blastocysts. The present real-time quantitative RT-PCR results showed that TGFA induced upregulation of survivin expression in blastocysts. Thus, the levels of survivin mRNA were associated with the occurrence of apoptosis in blastocysts. Together with the observation that survivin knockdown increased susceptibility to apoptotic stimuli in mouse preimplantation embryos [24], our data suggest that induction of apoptosis in singly cultured blastocysts is due to downregulation of survivin expression and, further, that expression of survivin is required for TGFA inhibition of apoptosis. The incidence of apoptosis and the levels of survivin mRNA in blastocysts treated with 0.1 pM of TGFA were comparable to those in blastocysts cultured in groups at a density of more than 30 embryos, suggesting that TGFA also may act as a autocrine factor for the inhibition of apoptosis and the upregulation of survivin. Since there is increasing evidence indicating that other growth factors (e.g., insulin-like growth factors and granulocyte-macrophage colony stimulating factor) also contribute in a paracrine and/or autocrine fashion to the regulation of apoptosis in mammalian preimplantation embryos [11, 12, 17, 18], the inhibition of apoptosis in group-cultured embryos could be mediated by other growth factors expressed by the embryo.

The effects of TGFA on apoptosis and regulation of survivin mRNA levels in blastocysts were most evident at 0.1 pM. The effective concentration of TGFA seems to be quite low because the activity of growth factors in preimplantation embryos is normally detected in the nanomolar or even micromolar range [11, 12, 17, 18]. However, a previous study using similar design of embryo culture system (numbers of embryos and size of culture drops) also showed same effective range of TGFA concentrations [19]. In addition, the rate of blastocoel expansion was stimulated by 0.1 pM of TGFA treatment [34]. Reduced activity of TGFA in high-dose-treated embryos was observed in our study and previous studies [19, 34]. Therefore, TGFA has a potential to act at very low concentrations in preimplantation embryos. In agreement with previous results showing lack of the effects of TGFA before the blastocyst stage [13, 34, 35], the effect of TGFA on upregulation of survivin mRNA was not evident before the eight-cell stage. Embryos may not have enough time to respond to TGFA treatment before this stage. Alternatively, the TGFA/PI3K/survivin pathway may be less functional before the blastocyst stage because of posttranscriptional regulation.

These data suggest that TGFA secreted from the maternal reproductive tract may maintain the sufficient amount of survivin for growing blastocysts in vivo in a paracrine manner. Since mouse and human fertilized oocytes and preimplantation embryos at all subsequent stages of development express both the TGFA ligand and its receptor themselves [11, 12], blastocysts cultured with neutralizing anti-TGFA antibody alone are likely to show an increase of apoptosis via a blockade of the autocrine effect of TGFA. However, in our study and in previous work [19], researchers failed to demonstrate such predicted effects. In fact, TGFA-null blastocysts show a higher incidence of apoptosis than wild-type embryos [23]. These results might be explained by the antibody's not penetrating into the ICM after compaction and tight junction formation [19].

Different anti- and pro-apoptotic genes are expressed during the development of the mammalian preimplantation embryo [17]. Among anti-apoptotic genes, survivin-null embryos stop developing at the early blastocyst stage in vitro and show embryonic lethality at the preimplantation period in vivo [36]. Thus, survivin-null embryos might not be able to survive receiving even essential growth factors, including TGFA. We used an antisense approach to suppress the expression of survivin in preimplantation embryos [24]. Apoptosis in blastocysts cultured singly was inhibited by TGFA treatment in both untreated and missense-treated groups. However, increased apoptosis by antisense oligonucleotides treatment in blastocysts was not suppressed by the TGFA treatment. These results strongly suggest that upregulation of survivin is responsible for the anti-apoptotic effect of TGFA in blastocysts.

Previous studies have shown that several growth factors inhibit apoptosis through activation of the PI3K or NF{kappa}B signaling pathways and that, subsequently, survivin expression is upregulated in both mouse and human cell types [37–44], with the exception of the endothelial cells, in which the IL-11/activator of the transcription 3 (STAT3) pathway regulates survivin [43]. In multiple myeloma cells, IGF-I upregulates survivin expression through the NF{kappa}B pathway [40], whereas this action of IGF-I is mediated through the PI3K pathway in thyroid carcinoma cells [41]. Thus, specific growth factors upregulate survivin expression via different signal transduction pathways in a cell-type-specific manner.

To gain further insight into downstream events in TGFA-mediated upregulation of survivin, we studied the involvement of PI3K and NF{kappa}B signaling pathways in mouse preimplantation embryos. Since activation of the STAT3 signaling pathway was reported to induce apoptosis in mouse blastocysts [45], the STAT3 pathways were excluded from this study. The results of the present study and previous studies [26] demonstrated that both PI3K and NF{kappa}B were expressed in mouse preimplantation embryos. However, since NF{kappa}B activation is observed only at the early one-cell stage [26] and NF{kappa}B inhibitors did not decrease levels of survivin mRNA in blastocysts, the NF{kappa}B signaling pathway appears to be present in early one-cell-stage embryos and no longer functional after the late one-cell stage. Conversely, PI3K inhibitors could block the paracrine effect of TGFA on upregulation of survivin mRNA in blastocysts, indicating involvement of a PI3K pathway in TGFA-induced upregulation of survivin. This selective signaling pathway may be specific to blastocysts in regulation of survivin expression by TGFA.

In the present study, in vitro treatment with TGFA suppressed apoptosis and increased the expression of survivin mRNA in blastocysts. However, under in vivo conditions, other members of EGF family known to bind EGFR (e.g., EGF, heparin-binding EGF, and amphiregulin) [13, 46, 47] may compensate for TGFA function. Thus, further studies will be required to fully elucidate the mechanisms of survivin regulation in mouse blastocysts in the maternal reproductive tract.

FOOTNOTES

1 Correspondence: Kazuhiro Kawamura, Department of Obstetrics and Gynecology, Akita University School of Medicine, Hondo 1–1–1, Akita 010-8543, Japan. FAX: 81 18 884 6447; kawamura{at}yf7.so-net.ne.jp Back

Received: 12 April 2005.

First decision: 6 May 2005.

Accepted: 25 July 2005.

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