Mol. Hum. Reprod. Advance Access originally published online on February 11, 2005
Molecular Human Reproduction 2005 11(3):183-188; doi:10.1093/molehr/gah136
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Expression of Smac/DIABLO in mouse preimplantation embryos and its correlation to apoptosis and fragmentation
Division of Obstetrics and Gynecology, Department of Reproductive and Developmental Medicine, Akita University School of Medicine, 1-1-1, Hondo, Akita 010-8543, Japan
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Akita University School of Medicine, 1-1-1, Hondo, Akita 010-8543, Japan. Email: fukudaj{at}obgyn.med.akita-u.ac.jp
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Abstract |
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Regulation of early embryonal development during fertilization and implantation is crucial for mammalian reproduction. Several studies have described cell death during preimplantation embryogenesis in a range of mammalian species, both in vivo and in vitro. Therefore, apoptosis may be involved in early embryonic arrest and the characteristic cytoplasmic fragments are the equivalents of apoptotic bodies, the end-product of apoptosis. Although apoptosis is expected to associate with fragmentation in early preimplantation embryos, the mechanism through which this fragmentation occurs has not been elucidated. Recently, second mitochondria-derived activator of caspase/Direct IAP Binding Protein with Low pI (Smac/DIABLO) was identified as a mitochondrial protein that is released into the cytosol during apoptosis. Once released, the Smac/DIABLO blocks the anti-apoptotic activity of inhibitor of apoptosis proteins (IAPs). We hypothesized that the Smac/DIABLO may be involved in the fragmentation of mouse preimplantation embryos. Therefore, we investigated the expression of Smac/DIABLO mRNA and protein and its localization in mouse oocytes and preimplantation embryos. Smac/DIABLO mRNA was detected by RT–PCR in the oocytes and the preimplantation embryos. Immunohistochemistry studies showed that the Smac/DIABLO protein localized in mitochondria and was released into the cytosol in both fragmented embryos and embryos in which apoptosis was induced by staurosporine. These observations indicate that the Smac/DIABLO is involved in the fragmentation and apoptosis of preimplantation embryos.
Key words: apoptosis/preimplantation embryo/Smac/DIABLO
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Introduction |
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The preimplantation stage of mammalian development corresponds to the period between ovulation and implantation and is crucial for reproductive success (Levy, 2001
). Recent studies have focused on the role of apoptosis in the degeneration of preimplantation embryos in vitro (Hardy, 1999, 2001). In the culture environment, apoptosis of early embryos has been ascribed to the lack of maternal factors, such as essential survival factors and growth factors and cytokines released by the maternal cells (Brison and Schultz, 1997; Hardy, 1999, 2001; Sjoblom et al., 1999; Spanos et al., 2000). Cell death has been observed during the preimplantation embryogenesis both in vivo and in vitro in a range of mammalian species (Hardy, 1997, 1999). Cells and nuclei exhibiting morphological features of apoptosis (Kerr et al., 1972; Wyllie et al., 1980), including cytoplasmic and nuclear fragmentation, chromatin condensation, DNA fragmentation, and phagocytosis, have been identified in human preimplantation embryos (Mohr and Trounson, 1982; Hardy et al., 1989, 1996, 1997, 1999; Jurisicova et al., 1996). These findings suggest that apoptosis may be involved in the early embryonic arrest and the observed cytoplasmic fragments are the equivalents of apoptotic bodies, the end-product of apoptosis (Jurisicova et al., 1996; Hardy, 1999). On the other hand, apoptosis does not occur before the blastocyst stage in normally developing mouse embryos, or compaction in normally developing human embryos (Handyside et al., 1986; Hardy, 1999). Similar observations in blastocysts of other species in vivo indicate that the apoptosis is playing a role in development at this stage. (Hardy, 1999)
Preimplantation embryo development is continuously regulated for the presence of both pro- and anti-apoptotic factors (Jurisicova et al., 1998; Warner et al., 1998a; Exley et al., 1999; Spanos et al., 2002). The inhibitor of apoptosis proteins (IAPs) family of genes has an evolutionarily conserved role in regulating programmed cell death in animals ranging from insects to humans (Clem and Miller, 1994; Hay et al., 1995; Rothe et al., 1995; Roy et al., 1995; Duckett et al., 1996; Uren et al., 1996). In mammals, the IAP family includes cIAP1, cIAP2, XIAP, Livin/KIAP, NAIP, BRUCE/Apollon, PIAP and Survivin. With the exception of the last four, which are thought to have cell cycle functions, these proteins bind to and inhibit both initiator caspases, such as caspase-9 and effector caspases, such as caspase-3 and caspase-7 (Deveraux et al., 1997, 1998; Roy et al., 1997; Kasof and Gomes, 2001; Silke and Vaux, 2001; Vucic et al, 2000). The IAPs are characterized by a novel domain of 
70 amino acids termed the baculoviral IAP repeat (BIR) (Crook et al., 1993; Birnbaum et al., 1994). The number of BIR domains ranges from one to three, which are all present in the N-terminal half of the protein, and are necessary for the anti-apoptotic activity of IAPs by virtue of their ability to bind and inhibit distinct caspases (Deveraux et al., 1997, 1998; Roy et al., 1997). For example, the XIAP is structurally characterized by three tandem repeats of the BIR domain at its N-terminus and a C-terminal RING finger domain. Its inhibitory activity is mediated through the BIR (BIR2) domain together with the immediately adjacent linker region, which is responsible for binding to and inhibiting active, processed caspase-3 and caspase-7 (Takahashi et al., 1998), whereas the third BIR (BIR3) domain is involved in interacting with and suppressing caspase-9 (Deveraux et al., 1999; Sun et al., 2000).
Recent studies have identified second mitochondria-derived activator of caspase/Direct IAP Binding Protein with Low pI (Smac/DIABLO) as a 239 amino acid precursor protein and a 29 kDa mitochondrial protein, which is processed into a 23 kDa mature protein that translocates to the cytosol after an apoptotic trigger (Chai et al., 2000; Du et al., 2000; Verhagen et al., 2000; Wu et al., 2000; van Loo et al., 2002). Smac/DIABLO normally resides within the mitochondria, but is released into the cytosol with cytochrome c upon cellular stress. Mature Smac/DIABLO was found to promote caspase activation by binding and neutralizing IAPs, including XIAP, cIAP-1, cIAP-2 and survivin (Verhagen et al.., 2000; Song et al., 2003). For example, a recognition motif at the amino terminus of mature Smac/DIABLO binds the BIR3 domain of XIAP. The same amino terminal sequence of Smac/DIABLO also forms a stable complex with the BIR2 domain of XIAP (van Loo et al., 2002).
Although the mechanism by which Smac/DIABLO acts to enhance apoptosis has been studied in detail, Smac/DIABLO functioning has not yet been studied in embryos. We investigated the role of Smac/DIABLO in the development of mouse preimplantation embryos by determining the temporal pattern of the Smac/DIABLO mRNA and protein expression in mouse oocytes and preimplantation embryos up to the hatched blastocyst stage by RT–PCR and immunohistochemistry.
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Materials and methods |
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Collection of mouse oocytes and preimplantation embryos
Female IVCS mice, aged 9 weeks (Institute for Animal Reproduction, Ibaragi, Japan), were ovulation inducted with a single intraperitoneal injection of 10 IU of pregnant mare's serum gonadotrophin (Sigma, St Louis, MO) followed 48 h later by 10 IU of HCG (Sigma). The 2-cell stage embryos were collected from the oviducts of the mated female mice 48–49 h after HCG injection. The 2-cell stage embryos were washed three times with M2 medium (Sigma). Groups of 10–15 randomly selected embryos were placed in 30 µl drops of human tubal fluid (HTF) medium covered by Oil for Embryo Culture (IS Japan Co., Saitama, Japan), and cultured at 37°C in 5% CO2. Unfertilized oocytes were obtained from oviducts of unplugged mice 18–20 h after HCG injection. Oocytes were immediately incubated in HTF medium containing 300 µg/ml hyaluronidase (Sigma, St Louis, MO) for 5 min at 37°C to remove cumulus cells. For RT–PCR analysis, 4-cell, 8-cell, morula, blastocyst and hatched blastocyst stage embryos were collected from cultured embryos in each microdrop at 51–52, 69–70, 91–92, 119–120 and 167–168 h after HCG injection. Fragmented embryos from the mated female mice were collected from the cultured embryos 119–120 h after HCG injection. A total of 15 oocytes and embryos were suspended in 20 µl of HTF medium and stored at –80°C until mRNA extraction. All procedures involving the care and use of animals were approved by the Animal Research Committee at Akita University School of Medicine.
RT–PCR
RT–PCR for oocytes and preimplantation embryos was performed according to previously reported methods (Kawamura et al., 2002, 2003a,b,c). Briefly, poly (A)+mRNA was isolated from 15 mouse oocytes or preimplantation embryos of each stage (2-cell, 4-cell, 8-cell, morula, blastocyst and hatched blastocyst), and each mRNA sample was reverse transcribed into cDNA. Exogenous rabbit 
-globin mRNA (Life Technologies, Inc., Rockville, MD) was added to each sample before mRNA extraction in order to evaluate the efficiency of mRNA extraction and the RT procedure. The amount of cDNA subjected to each PCR reaction was equivalent to the number of genomes (e.g. one 2-cell stage embryo or one quarter of an 8-cell stage embryo), so that each PCR product was derived from the same number of transcribing genomes. The primers for Smac/DIABLO were made based on published sequences (Du et al., 2000; Verhagen et al., 2000) as shown in Table I. The PCR was performed according to the conditions described in the legend of Table I. As a positive control, mouse placenta cDNA was amplified simultaneously. As a negative control, the specimen in which the mRNA was substituted for water was amplified. The PCR products were separated by 2% agarose gel electrophoresis (Agarose-LE, Nacalai Tesque, Inc., Kyoto, Japan) in the presence of ethidium bromide (Sigma), and visualized with an UV transilluminator (Funakoshi, Tokyo, Japan). To confirm identity, bands of each PCR product were eluted from agarose gel using the QIAquick gel extraction kit (Qiagen KK, Tokyo, Japan), ligated into the pDrive Cloning vector (Qiagen KK), and cloned in accordance to standard protocols. Plasmid DNA was recovered using a Quantum Prep Plasmid Miniprep kit (Bio-Rad, Hercules, CA), cycle sequenced, and analysed in an ABI 100 DNA sequencer (PE Applied Biosystems, Tokyo, Japan) using T7 or SP6 site-specific primers.
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Immunohistochemistry
Oocytes, 2-cell, 4-cell, 8-cell and hatched blastocyst stage embryos were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at 4°C after thorough washing in 1% PBS–bovine serum albumin (BSA) (Sigma). The embryos were then permeabilized in PBS/0.1% Tween 20 (Sigma) for 5 min at room temperature. After blocking for 30 min at room temperature in 10% normal goat serum (DAKO Corp., Kyoto, Japan), the embryos were incubated with either 0.2 µg/ml rabbit anti-mouse Smac/DIABLO antibody (Chemicon, Temecula, CA) or 2.5 µg/ml fluorescein isothiocyanate-conjugated anti-mouse cytochrome c antibody (Bioscience) in 1% PBS–BSA, overnight at 4°C. After three washes in PBS, the embryos stained for cytochrome c were transferred to a drop of PBS on a slide and analysed under an epifluorescence microscope (Olympus Corp., Tokyo, Japan). Smac/DIABLO stained embryos were incubated with 1.0 µg/ml of goat anti-rabbit Arexa 488 fluorescein antibody (Molecular Probes) in 1% PBS–BSA, for 1 h at room temperature in the dark. After three washes in PBS, the embryos were transferred to a drop of PBS on a slide and analysed under an epifluorescence microscope. For negative controls, sections were subjected to the same method, except that the primary antibodies were replaced by the same concentration of rabbit immunoglobulin G (DAKO Corp.).
Induction of apoptosis by staurosporine
The 2-cell stage embryos were recovered from IVCS female mice as described above and pooled in M2 medium. Randomly selected groups of 10–15 embryos were placed in 30 µl drops of the HTF medium with 10 µM staurosporine. The embryos in which apoptosis was induced were collected 84–85 h after HCG injection.
JC-1 and Hoechst staining
The mitochondrial membrane potential was measured using the MitoTag JC-1 Assay kit (Intergen Company, NY) and Hoechst 33342 (Intergen Company, NY) according to manufacture's protocol. Oocytes and embryos were incubated with 0.2 µg/ml of JC-1 reagent and 1 µg/ml of Hoechst 33342 for 15 min at 37°C in 5% CO2 in air. After three washes in PBS, the embryos were transferred to a slide and analysed under an epifluorescence microscope.
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Temporal expression of Smac/DIABLO mRNA in mouse oocytes and preimplantation embryos
RT–PCR was performed to detect the Smac/DIABLO mRNA in mouse oocytes and preimplantation embryos at different stages (2-cell, 4-cell, 8-cell, morula, blastocyst and hatched blastocyst). Smac/DIABLO mRNA was detected in all stages as a 397 bp fragment (Figure 1). As an internal control,
-actin was detected by RT–PCR in the same samples. No significant differences were observed in the intensities of -globin amplification products. Following RT, PCR was performed three times on five separate pools of 15 oocytes and embryos at each stage, and the results were found to be reproducible.
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x174-HaeIII digest; posi: positive control=mouse placenta cDNA; neg: negative control=without template cDNA; bp: base pair.Expression of Smac/DIABLO and cytochrome c proteins in mouse oocytes and preimplantation embryos
Smac/DIABLO immunoreactivity was observed from the oocyte through the hatched blastocyst stage (Figure 2). These results were consistent with the expression of Smac/DIABLO mRNA. Both Smac/DIABLO and cytochrome c show the punctuate staining characteristic of mitochondrial localization in the normal embryo.
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Loss of mitochondrial membrane potential in fragmented embryos
JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide) is a lipophilic cationic dye that enters the inner mitochondrial matrix in its monomeric form when the mitochondrial membrane is polarized (Reers et al., 1991
, 1995). When the mitochondrion has a high mitochondrial membrane potential, the dye crosses the membrane and forms J-aggregates, which appear red under UV light. If the potential is low, the dye remains in its monomeric form and is fluorescent green (Cossarizza et al., 1993). In normal 2-cell embryos, the JC-1 labelling results in red mitochondrial staining, as is typical for non-apoptotic cells (Figure 3A). In fragmented embryos, the JC-1 labelling results in green cytoplasmatic staining, indicating loss of mitochondrial membrane potential, as is typical for apoptopic cells (Figure 3B). Using Hoechst staining, normal 2-cell embryos showed a non-apoptotic morphology (Figure 3C), whereas nuclear condensation and apoptotic bodies were observed in the fragmented embryos (Figure 3D).
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Release of Smac/DIABLO and cytochrome c from mitochondria into the cytosol in apoptosis-induced and fragmented embryos
Smac/DIABLO and cytochrome c showed diffuse staining of the cytoplasm in apoptosis-induced and fragmented embryos. These embryos were characterized by a loss of mitochondrial membrane potential (JC-1 staining) and abnormal nuclei (Hoechst staining) (Figure 4).
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Discussion |
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In this study we demonstrated for the first time the expression of Smac/DIABLO in mouse oocytes and preimplantation embryos. Smac/DIABLO mRNA and protein were expressed throughout the developmental stages of preimplantation embryos and in oocytes. Both Smac/DIABLO and cytochrome c were localized consistently in the mitochondria of normal embryos. In fragmented embryos or embryos in which apoptotis was induced by staurosporine, translocation of the Smac/DIABLO from mitochondria into the cytosol was observed by immunohistochemistry.
Apoptosis is mainly orchestrated by a family of aspartate-specific cysteine proteases known as caspases. Caspases are generally divided into two categories, the initiator caspases, which include caspase-2, -8, -9, and effector caspases, such as caspase-3, -6, and -7. A pro-apoptotic signal culminates following activation of an initiator caspase, which, in turn, activates effector caspases (Thornberry and Lazebnik, 1998; Shi, 2002). There are two well-characterized signal pathways leading to the activation of caspases: the death receptor pathway and the mitochondrial pathway. In the mitochondrial pathway, the Bcl-2 protein family, whose members may be apoptotic or pro-apoptotic, regulates cell death by controlling mitochondrial membrane permeability during apoptosis. Death signals induce the release of cytochrome c from mitochondria into the cytosol and the assembly of an apoptosome consisting of cytochrome c, adapter protein Apaf-1, and procaspase-9, triggering activation of caspase-9, which activates the effector caspases, such as caspase-3, resulting in cleavage of the broad spectrum of cellular targets and, ultimately, apoptosis (Green and Reed, 1998).
In this study, both Smac/DIABLO and cytochrome c localized consistently in mitochondria throughout the developmental stages of embryos. This result was expected, because apoptosis is not detected during normal development of early preimplantation embryos (Raff et al., 1993; Wei et al., 1996; Hardy, 1997, 1999). In contrast, in fragmented embryos and in embryos in which apoptosis was induced by staurosporine, translocation of Smac/DIABLO and low potential of mitochondrial membrane was observed. The pro-apoptotic drug staurosporine is a broad spectrum inhibitor of protein kinases and is known to induce apoptosis in preimplantation embryos (Warner et al., 1998b). Staurosporine does not require the presence of apoptosis-inducing factor (AIF) to cause cell death (Joza et al., 2001), but requires the presence of either Bax or Bak to induce mitochondrial release of cytochrome c and cell death (Weil et al., 2001). Like cytochrome c, it has been reported that the Smac/DIABLO is also released into the cytosol in response to some apoptotic stimuli, as was shown previously in different cell types by immunohistochemistry and Western blot analysis (Du et al., 2000; Verhagen et al., 2000; Fu et al., 2003; Kandasamy et al., 2003; Song et al., 2003). We demonstrated that the Smac/DIABLO protein localized in mitochondria of normal embryos and that the protein was released from mitochondria into the cytosol in either fragmented embryos or apoptotic embryos following induction by staurosporine, confirming the previously described results. However, we were unable to confirm the translocation of Smac/DIABLO in apoptotic embryos by Western blot analysis, because of the limited amount of protein available from mouse embryos.
In summary, the Smac/DIABLO is expressed throughout the developmental stages of preimplantation embryos and in oocytes. The Smac/DIABLO was released from mitochondria into the cytosol in fragmented and apoptotic embryos. Recently, we have reported that the IAP family members XIAP, cIAP-1, cIAP-2 and survivin were also expressed during early embryonic development (Kawamura et al., 2003c). IAP family members are essential anti-apoptotic genes expressed in preimplantation embryos, which may protect the embryos from apoptosis by inhibiting an apoptotic pathway involving caspases. Smac/DIABLO promotes caspase activity by binding to IAPs (Verhagen et al., 2000). Our observations indicate that these pro- and anti-apoptotic regulating systems co-exist during early embryonal development. However, further studies are necessary to understand exactly how the Smac/DIABLO is involved in the mechanism of fragmentation and apoptosis in preimplantation embryos.
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Acknowledgements |
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This work was supported by a Grant-in-Aid Scientific Research (C: 14571535) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
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Submitted on January 7, 2004; resubmitted on November 11, 2004; accepted on November 15, 2004.
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