Molecular Human Reproduction, Vol. 9, No. 3, 133-141,
March 2003
© 2003 European Society of Human Reproduction and Embryology
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Expression of apoptosis-related genes during human preimplantation embryo development: potential roles for the Harakiri gene product and Caspase-3 in blastomere fragmentation
Submitted on April 4, 2002; resubmitted on August 14, 2002. accepted on November 26, 2002
1 Division of Reproductive Sciences, Departments of Obstetrics and Gynecology and 2 Department of Zoology, University of Toronto, Toronto, Ontario, Canada and 3 Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02114, USA
4 To whom correspondence should be addressed at: Samuel Lunenfeld Research Institute, Room 876, 600 University Avenue, Toronto, Ontario, M5G 1X5, Canada. e-mail: jurisicova{at}mshri.on.ca
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In order to resolve the mechanisms and reasons of cellular fragmentation it is crucial to understand what genes may be responsible for regulation of this process. We report herein that human oocytes and preimplantation embryos possess abundant levels of transcripts encoding cell death suppressors, Mcl-1, Bcl-x and Bag-1, and the cell death inducer genes, Bax and Caspase-2. Lower but detectable levels of mRNA expression for the Bfl-1/a1, Bcl-w, Harakiri (Hrk) and Caspase-3 genes were also detected during all developmental stages. We also performed analysis of gene expression in single human embryos exhibiting various degrees of fragmentation at the 2-, 4- and 8-cell stages. At the 4-cell stage, embryos displaying 30–50% fragmentation showed a significant increase in Hrk mRNA levels (P = 0.016). Immunostaining with anti-Hrk antibody confirmed increased staining in some, but not all, fragmented embryos. While Caspase-3 transcripts were elevated in both 4- and 8-cell embryos exhibiting a severe degree of fragmentation, this difference did not reach statistical significance. However, accumulation of Caspase-3 mRNA in fragmented embryos was paralleled by an induction of Caspase-3-like activity. These findings suggest that cellular fragmentation in a subset of human preimplantation embryos could be regulated by certain components of a genetic programme of cell death.
Key words: apoptosis/Caspase-3/cell death/embryo/Hrk
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Introduction |
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With improved protocols for hormonal stimulation, more defined culture conditions, and improved transfer techniques, several groups have reported implantation rates in the range of 15–23% for good quality human embryos conceived in vitro (Dawson et al., 1995; Giorgetti et al., 1995; Ebner et al., 2001). However, the overwhelming majority of human preimplantation embryos still fail to establish viable pregnancies due to various kinds of cellular and morphological abnormalities. The most frequently observed defects during preimplantation development are alterations in nuclear-to-cytoplasmic ratios manifested as multinucleation (Tesarik et al., 1988; Winston et al., 1991; Hardy et al., 1993) as well as organelle and other cytoplasmic dysmorphisms (Van Blerkom et al., 2000). Such abnormalities interfere with the developmental potential of embryos and are reflected in the outcome of IVF procedures, since low implantation rates and high embryo loss was observed upon transfer of such embryos (Balakier and Cadesky, 1997; Meriano et al., 2001). Embryo fragmentation remains the most common cellular anomaly observed in almost all patients undergoing IVF. Human embryos with <20% fragmentation exhibit similar implantation rates comparable to that of embryos with normal (non-fragmented) morphology. This is contrasted by marked reduction of the implantation rate in embryos with a substantial amount (30–50%) of cellular fragmentation (Ziebe et al., 1997; Ebner et al., 2001). While the implantation rate of extensively fragmented embryos is low, these embryos do yield relatively high (18 versus 57%) biochemical pregnancy rates (Ziebe et al., 1997), suggesting that these embryos fail to proceed through peri- and/or post-implantation development. However, recent studies performed on day 3 embryos reported that the pattern of fragmentation, rather than degree, is a more reliable predictor of developmental potential (Alikani et al., 1999).
The origin as well as the mechanisms of how and why fragmentation in embryos occurs is still unclear. While some patterns of fragmentation had been attributed to a process of oncosis (Van Blerkom et al., 2001), a series of experiments performed in our laboratory led us to the hypothesis that a subset of embryo fragmentation may be a result of apoptotic-like disintegration. These experiments revealed that many cellular hallmarks, including nuclear and cytoplasmic condensation as well as DNA cleavage, are present frequently, but not always, in arrested-fragmented human embryos (Jurisicova et al., 1996). These findings generated some controversy regarding whether embryo fragmentation is or is not associated with activation of an apoptotic cascade (reviewed in Hardy, 1999). Cell death consistent with apoptosis, especially in the inner cell mass (ICM) lineage, occurs in blastocysts of many different mammalian species (El-Shershaby and Hinchliffe, 1974; Mohr and Trounson, 1982). Primate blastocysts, including those of humans, eliminate 
20% of their cells at day 7 post-insemination (Hardy et al., 1989). This induction of cell death occurs in both the ICM and in the trophectodermal lineage (Hardy, 1997; Jurisicova et al., 1999). In addition, human embryos frequently arrest and undergo fragmentation at the 4–8-cell stage (although fragmentation at the 2-cell stage has been observed as well; Antczak and Van Blerkom, 1999), when it is believed that transition from the maternal to embryonic genome occurs (Braude et al., 1988).
Programmed cell death (PCD) is a precisely coordinated event dependent upon the actions and interactions of a number of gene products that either suppress or activate the process of cellular self-destruction (for review, see Adams and Cory, 1998; Vaux and Korsmeyer, 1999). Currently, there are >100 different genes whose expression affects cell survival. While functional importance for several genes had been evaluated in oocytes (Tilly, 2001), it is unclear if any of these are involved in the regulation of cellular fragmentation during the preimplantation period of development. In the present study, we designed experiments to establish the developmental expression profile of a subset of cell death suppressors (Bcl-x, Mcl-1, Bcl-w, Bfl-1/a1, Bag-1) and cell death inducers [Bax, Harakiri (Hrk)] and caspases (Caspase-1, -2 and -3) in human preimplantation embryos. The selection of genes chosen for analysis was based on earlier reports of their expression in mouse preimplantation embryos (Jurisicova et al., 1998b; Exley et al., 1999) or ovary (reviewed in Tilly, 2001) and placenta (Huppertz et al., 1998). The ovarian expression of some of these genes may reflect a contribution of the oocyte, whereas expression in placenta may reflect contribution of trophoblast lineage established at the blastocyst stage. We compared embryos that would be selected for uterine transfer in IVF with a low level of fragmentation (<20%) and embryos exhibiting extensive (30–50%) or complete (100%) fragmentation. Changes in the accumulation of mRNA transcripts encoded by these cell death regulatory genes were analysed and compared in embryos of different categories.
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IVF and embryo culture
Spare human uninseminated oocytes and preimplantation embryos were obtained from the IVF Program in the Division of Reproductive Sciences at the Toronto General Hospital or from the Toronto Center for Advanced Reproductive Technologies. Patients who chose not to freeze their embryos for future transfers were asked to donate them for research and informed consent was obtained. The Human Ethics Committee of the Toronto General Hospital and the University of Toronto approved this research.
Ovarian stimulation was carried out using a GnRH agonist (Lupron; Abbott Pharmaceuticals, Montreal, Canada) in a long protocol (Greenblatt et al., 1995), and hMG (Pergonal, Serono Canada, Oakville, Ontario; or Humegon, Organon Canada, Scarborough, Ontario) or hFSH (Fertinorm or Gonal-F, Serono; and Puregon, Organon). IVF and ICSI were performed using standard techniques as previously described (Segal and Casper, 1992; Casper et al., 1996).
Spare embryos of variable quality that appeared to arise from normally fertilized oocytes with two pronuclei were used for subsequent analysis. Embryos were cultured in human tubal fluid (HTF) medium (Irvine Scientific, Santa Ana, CA, USA) supplemented with 10% synthetic serum substitute (Irvine Scientific) at 37°C in an incubator with 5% CO2 and 95% air. Assessment of embryo quality with respect to fragmentation and developmental stage was recorded daily. The proportion of cytoplasmic volume effected by fragmentation as a percentage was used to cluster embryos into subcategories. Immature oocytes prior to germinal vesicle breakdown (GV stage) or at metaphase I (MI) were obtained from patients undergoing the ICSI procedure. These were denuded of cumulus cells with hyaluronidase and were either used immediately (GV stage) or matured in vitro for 24 h until they reached metaphase II (MII) (n = 5). An additional five mature MII oocytes were obtained from a patient, whose husband failed to produce a sperm sample. In all cases, oocytes were never exposed to sperm and the zona pellucida was removed from all samples by brief exposure to acid Tyrode’s (pH 2.5) to prevent possible contamination with sperm nucleic acids.
Expression of cell death regulatory genes during preimplantation embryo development
Gene expression was determined by a quantitative RT–PCR followed by Southern dot blot analysis (QADB) assay detailed previously (Rambhatla et al., 1995; Jurisicova et al., 1998b, 1999). Briefly, cDNA derived from a single oocyte or embryo at each developmental stage were amplified as described (Brady and Iscove, 1993). Each developmental stage was represented by five to eight individual samples. The amplified material was then analysed by hybridization of dot blots with cDNA probes radiolabelled by random-priming, followed by quantification of signals using a phosphorimager and a calibrated [14C] microscale (Amersham, Baie d’Urfe, Quebec, Canada) for correcting times of exposure (Rambhatla et el., 1995). Pre-screening with EF1
was carried out to assess the quality of cDNA amplification (expression of EF1 does not change markedly during the 2–8-cell stage; Wang and Latham, 2000) The cDNA probes used for analysis recognized the 3'-untranslated regions of target genes. The probes used were either donated (Bcl-w, Bfl-1/a1, Hrk, casp-3) or were cloned from an oligo-dT-primed human full-term placenta (ATCC, Rockville, MD, USA) or teratocarcinoma cDNA libraries (Bcl-x, Bag-1, casp-1, casp-2, EF1). The identities of the cDNA clones were confirmed by sequence analysis using a Sequenase version 2.0 DNA kit (USB, Cleveland, OH, USA). Probes for Mcl-1 and Bax were obtained by RT–PCR of RNA isolated from pre-menopausal adult human ovary, using synthesized primers based on published sequences (see Table I). Identities of the respective 600 and 500 bp amplified DNA fragments were confirmed by sequence analysis using a dsDNA Cycle Sequencing Kit (Life Technologies, Burlington, Ontario, Canada).
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Detection of alternative splicing forms, Bcl-xL and Bcl-xS, in fragmented human embryos
In order to determine which form of Bcl-x is expressed by human preimplantation embryos, we performed RT–PCR with specific primers (Table I spanning the alternative splice site of the Bcl-x gene) (Boise et al., 1993). Briefly, 8–10 extensively fragmented 4–8-cell stage embryos from seven patients were lysed in 50 µl of guanidinium isothiocyanate solution (Brady and Iscove, 1993). Total nucleic acid was recovered by ethanol precipitation using glycogen as a carrier, and RT–PCR was then performed as previously described (Jurisicova et al., 1998b). Each PCR cycle consisted of denaturation at 95°C for 60 s, annealing at 58°C for 60 s, and extension at 72°C for 90 s. The expected sizes for the amplified products were 760 bp for Bcl-xL and 550 bp for Bcl-xS. To confirm the identity of amplified products, Southern blot analysis was performed with a radiolabelled cDNA probe recognizing the shared coding region of both Bcl-x splice variants. The obtained signal was then analysed by quantification of signals using a phosphorimager and expressed as ratio of Bcl-xS/L (density of lower/higher band).
Immunolocalization of Hrk
Twenty-seven human embryos between 4- and 10-cell stage were fixed for 10 min in 10% buffered formalin, transferred on slides, air-dried and stored at –20°C. Upon defrosting, slides were washed in phosphate-buffered saline (PBS) and antigen retrieval was performed in a microwave (3x3 min) in sodium citrate buffer (pH 6.0). Subsequently, slides were rinsed in PBS and blocked in 10% goat serum in PBS with 0.05% Triton X. Affinity-purified rabbit anti-human Hrk polyclonal antibody (Sanz et al., 2000) or Hrk N20 goat anti-human polyclonal antibody (sc-6972; Santa Cruz Biotechnology Corp., Santa Cruz, CA, USA) diluted 1:100 in PBS supplemented with host serum were applied to samples and incubated overnight at 4°C. Upon further washing and incubation with dilution (1:200) of secondary biotinylated antibody (Vector Laboratories, Burlingame, CA, USA), final labelling was performed with streptavidin Texas Red (Calbiochem, San Diego, CA, USA). For subcellular localization of Hrk, dual labelling was performed using an antibody to anti-human mitochondria (MAB1273; Chemicon, Temecula, CA, USA) with goat anti-mouse Cy2 (Molecular Probes, OR, USA) secondary antibody. Samples were washed, counterstained with the DNA-binding fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI; Sigma Chemical Co., St Louis, MO, USA) and viewed under a deconvolution microscope (Olympus IX70). Embryos were serially scanned and optical sections (1 µm) were analysed using DeltaVision software (Applied Precision Inc., CA, USA). All samples were processed and analysed at the same time to avoid variability caused by decay of fluorescence.
Single cell caspase activity assay
Caspase activity was assessed in arrested embryos without or with fragmentation as previously described (Perez et al., 1999). This single cell fluorescent assay reflects predominantly the activity of caspase-3 and -7, as determined by trapping of fluorochrome-conjugated caspase substrate [aspartic acid, glutamic acid, valine, aspartic acid (DEVD–Asp-Glu-Val-Asp)] within a cell. Briefly, embryos were cultured in the presence of rhodamine-conjugated substrate (PhiPhiLux; OncoImmunin Inc., College Park, MD, USA) for 3 h. Subsequently, samples were washed three times, fixed in 4% paraformaldehyde, transferred onto slides and stained with DAPI, as previously described (Jurisicova et al., 1998a). This approach permitted simultaneous analysis of chromatin status (diffuse or condensed), the distribution of DNA within blastomeres and fragments, and caspase-3-like activity. Embryos were viewed by fluorescent microscopy using appropriate filters, and representative photomicrographs were taken.
Statistical analysis
Analysis of potential differences in transcript levels between embryos with 0–20% and 30–50% fragmentation was performed by Student’s t-test and analysis of variance using the SigmaStat statistical package (Version 1.0).
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Sample characteristics
We obtained 45 uninseminated human oocytes and 101 spare preimplantation embryos at different stages of development. These oocytes and embryos were processed as described and pre-screened for the quality of amplified cDNA by hybridization with elongation factor-1
(EF1). Ninety-nine samples (68%) showed detectable hybridization signal. The rate of failure of amplification/and or hybridization was 27 and 35% for embryos without or with <20% fragmentation respectively. From this pre-screened pool, 20 oocytes and 30 embryos at different developmental stages were selected for this study based on strong EF1 hybridization signals (data not shown). This pre-screening process allowed the analysis of 5–8 oocytes or embryos at each developmental stage. For embryos in the <20% fragmented category the population at each stage comprised: 2 cells, 3x0%, 1x10%, 1x15%; 4 cells, 3x0%, 1x5%, 1x10%; 8 cells, 2x5%, 2x10%, 1x15%.
Expression of genes that regulate PCD in embryos with <20% fragmentation
Polyadenylated mRNA transcripts encoded by the Bfl-1/a1, Bcl-x, Mcl-1 and Bag-1 genes were detected in GV and MII stage unfertilized oocytes, as well as throughout preimplantation embryonic development (Figure 1). Oocytes expressed a low level of Bcl-w (data not shown) that became undetectable at the 2-cell stage. The first embryonic stage that had a detectable level of Bcl-w transcript was the morula, and expression of this gene persisted to the blastocyst stage, although it remained constantly low. Expression of Bfl-1 (Figure 1A) and Bcl-x (Figure 1B) were easily detected during early embryonic stages, but their transcript accumulation was only 10–20% of the levels of Mcl-1 (Figure 1C) and Bag-1 (Figure 1D) expression.
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Transcripts for Bax and Hrk, two pro-apoptotic members of the Bcl-2 family, were detected in both immature and mature oocytes and were consistently expressed in embryos during all developmental stages. Levels of Bax mRNA (Figure 2A) were higher than those of Hrk mRNA (Figure 2B).
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Analysis of caspase expression during preimplantation development revealed the presence of high levels of Caspase-2 (Figure 2C) and Caspase-3 (Figure 2D) transcripts, but no detectable expression of the Caspase-1 gene (data not shown). The levels of Caspase-2 mRNA showed an accumulation from immature oocytes to the blastocyst stage. In contrast to Caspase-2 expression, Caspase-3 expression was most abundant in GV stage oocytes. Expression of the Caspase-3 gene could be detected at all embryonic stages, but the mRNA was present in lower quantities than the Caspase-2 transcript.
Expression of PCD regulatory genes in embryos undergoing fragmentation
Based on the apparent trends observed for six genes detected in human non-fragmented embryos (Bag-1, Bcl-x, Mcl-1, Hrk, Caspase-2 and Caspase-3), we analysed transcript accumulation in embryos undergoing fragmentation between the 2- and 8-cell stage. We compared 15 embryos with a low degree of fragmentation (<20%) and 20 embryos with partial (30–50%) or complete fragmentation. Similarly, pre-screening with EF1a was used for assessing quality of cDNA amplification and complete amplification/hybridization failure was observed in 44/98 (45%) for partially and 25/51 (49%) for completely fragmented embryos respectively. At each developmental stage, embryos were split into subgroups of five embryos with 0–20% fragmentation and 30–50% fragmentation. One separate category consisted of five embryos that were completely fragmented (100%) late on day 2/early day 3 post-insemination and thus were difficult to assign into a specific developmental category (i.e. 4- or 8-cell).
All six genes were expressed in each subgroup of embryos. While the overall trend of expression for Hrk, Bag-1 and Caspase 3 remained the same between the first (described above) and second (current) in embryos with <20% of fragmentation, total transcript levels for Bcl-x, Mcl-1 and Caspase-2 were variable. We observed several trends towards changes in the transcript accumulation that appeared stage specific, but these did not reach statistical difference (e.g. Bag-1, Mcl-1 and Caspase-3). Interestingly, the only significantly up-regulated transcript in embryos with 30–50% versus 0–20% fragmentation was that of Hrk (Figure 3), and this occurred at the 4-cell stage (P = 0.016). No differences were observed in transcript levels for Caspase-2, nor in total levels of Bcl-x at any developmental stage regardless of the degree of embryo fragmentation. Since QADB assay does not distinguish between the long (death-suppressing) and short (death-promoting) splice variants of Bcl-x, the possible contribution of alternate splicing of the Bcl-x gene was directly tested in another set of experiments (see below).
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Alternative splicing of Bcl-x in embryos with >30% fragmentation
Using primers that span the alternative splicing site within the Bcl-x transcript, we simultaneously analysed expression of Bcl-xL and Bcl-xS transcripts. Only fragmented embryos were analysed since we were not able to obtain sufficient numbers of good quality embryos from the same patients. Nucleic acids from four pools of fragmented embryos resulted in the amplification of both Bcl-xL and Bcl-xS transcripts with variable ratios (Figure 4). The relative balance of the short-to-long Bcl-x splice variants favoured the short isoform (3–4-fold higher) in two groups of embryos analysed (lanes 1 and 4), while ratios in other two groups (lanes 2 and 3) were favouring Bcl-xL. Thus, even though fragmented embryos do not alter expression of total Bcl-x content, in some cases they appear to regulate alternative splicing of the Bcl-x gene in favour of Bcl-xS. Three pools of embryos did not produce any detectable product suggesting that their RNA was already degraded at the time of harvesting. This is consistent with our previous findings observed in mouse fragmented embryos (Jurisicova et al., 1998b).
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Hrk immunolocalization
Since we observed up-regulation of Hrk mRNA in fragmented embryos, we decided to assess protein levels of this Bcl-2 family member. Twenty-seven embryos were analysed using indirect immunocytochemistry and representative results are shown in Figure 5. Five embryos at the 4–10-cell stage showing no signs of fragmentation had low to undetectable immunostaining (Figure 5B), comparable to control stained embryos (Figure 5A). The remaining 22 embryos showed various degrees of fragmentation between 30 and 100% and displayed variable levels of Hrk from non-detectable to high (Figure 5C–E). The degree of fragmentation did not correlate with levels of Hrk protein. The same trend and pattern of staining was observed with both anti-rabbit and anti-goat Hrk antibody used in this study (data not shown). Subcellular localization of Hrk appeared granular with increased perinuclear clustering suggestive of intracellular organelle localization. This pattern of staining is identical to that previously described for localization of epitope-tagged Hrk protein (Inohara et al., 1997). In order to establish whether Hrk may localize to mitochondria, we performed dual labelling immunocytochemistry. As can been seen in Figure 5F, Hrk did not localize to mitochondria, except for a few areas highlighted by the arrows (Figure 5G). However, at the blastocyst stage (Figure 5H), Hrk protein was detected in cells of the ICM area which appeared healthy, but not in the mural trophectoderm and revealed diffuse cytoplasmic localization.
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Caspase-3 activity in human embryos
Analysis of Caspase-3-like activity was performed on 25 embryos and representative results are shown in Figure 6. In general, caspase activity was observed in extensively fragmented (50–100%) embryos (in 10/12 embryos), although it appeared to be restricted only to fragments and a few blastomeres that contained chromatin (Figure 6C–F). Frequently, DNA within these fragments exhibited signs of condensation and fragmentation. By comparison, less intense fluorescence resulting from cleavage of the Caspase-3-like substrate was observed in two of eight embryos with 30% of fragmentation. Lastly, caspase activity was absent in fragments without chromatin and was not detectable (0/5) in blastomeres of non-fragmented embryos (Figure 6A, B). However, the polar body, which is known to undergo cell death independent of the health status of the embryo, was positive for Caspase-3-like activity.
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Discussion |
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Identification of PCD as one of the possible mechanisms underlying certain types of embryo fragmentation has opened a new avenue of investigation of the molecular and genetic events controlling early embryonic decisions. Based on our previous work and the work of several other groups (Yang et al., 1998; Alikani et al., 1999; Antczak and Van Blerkom, 1999) it is becoming evident that there is more than one kind of cellular fragmentation. While some embryos exhibit fragmentation associated with a loss of blastomeres, other embryos fragment without the loss of blastomeres. Whether the molecules regulating classical cell death pathways are also involved in the regulation of both kinds of cytoplasmic fragmentation remains to be determined. This current study attempts to correlate the expression of various genes in embryos with different proportions of cellular fragmentations, irrespective of fragmentation subtype. In the future it would be informative to perform gene expression studies in embryos exhibiting different subtypes of fragmentation and compare their molecular signatures.
Analysis of the patterns of cell death gene expression during mouse preimplantation embryo development revealed that oocytes and early embryos express transcripts encoded by many genes involved in PCD activation and execution (Jurisicova et al., 1998b; Exley et al., 1999). Murine embryos, which undergo complete fragmentation at the 1-cell stage, not only contained low levels of mRNA for cell death suppressors, but also appeared to have augmented maternal stores of certain genes that promote PCD. Results from the experiments described in this current report revealed a similar expression profile for several PCD-associated genes in human and mouse embryos, suggesting that the mechanism underlying embryo fate may be evolutionarily conserved. However, it is unlikely that the presence or absence of a single protein will determine the survival or death outcome in preimplantation embryos because most cell death gene products work through a complex network of homo- and heterodimerization. Working on this premise, we also compared ratios between Bcl-x/Hrk, Mcl-1/Hrk, Bag-1/Hrk, Bcl-x/Bag-1 and Mcl-1/Bag-1. None of these ratios, however, showed significant differences between different proportions of fragmentation (data not shown).
Similar to murine embryos, Bcl-x is expressed in human embryos during preimplantation development, and also undergoes alternative splicing, creating two proteins: Bcl-xS, which lacks two typical Bcl-2 homology domains termed BH1 and BH2, and Bcl-xL, which retains these domains (Boise et al.,1993). Bcl-xL is a very potent cell death suppressor, while Bcl-xS renders cells more susceptible to pro-apoptotic signals, even in the face of concomitant expression of Bcl-xL (Heermeier et al., 1996; Minn et al., 1996). In normal mouse embryos, Bcl-x expression is entirely attributable to the protective Bcl-xL isoform. However, Bcl-xS transcripts are detected in a subset of murine fragmented embryos (Jurisicova et al., 1998b). The data presented here from human fragmented embryos suggest that changes in Bcl-x splicing, leading to a shift in the ratio of the long-to-short isoform, may be one of the factors involved in the regulation of embryo demise.
Human oocytes and embryos also expressed the cell death inducers, Bax and Hrk. The proteins encoded by these two genes bind Bcl-xL, but not Bcl-xS, with high affinity (Inohara et al., 1997). Hrk, a member of the Bcl-2 family of proteins containing only the BH3 domain, is transcriptionally regulated in various cell types (Imaizumi et al., 1999; Sanz et al., 2000), being induced within an hour after administration of cell death stimuli (Harris and Johnson, 2001). Our results indicate that extensively fragmented human embryos contain significantly higher transcript levels for Hrk at the 4-cell stage. Hrk immunolocalization confirmed increased accumulation of this protein in some fragmented embryos. Interestingly, severely fragmented embryos expressed lower levels of Hrk protein than less fragmented embryos, consistent with the expression pattern of other cell death inducers such as Bax, which are expressed for a short period of time when a cell makes a decision to die (Matikainen et al., 2002). Thus, it is possible that embryos activate PCD when Hrk levels begin to accumulate, particularly in an intracellular environment that may also be subjected to dwindling Bcl-xL levels.
Caspases, a growing family of cysteine aspartic acid-specific proteases, appear to function downstream of Bcl-2 family members to complete the programme of cell death (Fraser and Evan, 1996; Thornberry and Lazebnik, 1998). Transcripts for Caspase-2 and Caspase-3 were detected at all cleavage stages during human preimplantation embryo development. While levels of Caspase-2 mRNA were not altered during fragmentation, a trend towards increased accumulation of Caspase-3 mRNA was noted in human fragmented embryos, paralleled by induction of Caspase-3-like enzyme activity, but only in nucleated fragments. Recently, Martinez et al. (2002) reported the presence of activated caspases (using FITC–VAD–FMK substrate) in a majority of cellular fragments, but not in nucleated cells. Moreover, cellular fragmentation accompanied by caspase activity was observed in isolated blastomeres, which subsequently cleaved. Based on these observations, it was concluded that cellular fragmentation is accompanied by caspase activation, but it is not associated with apoptosis manifested by complete disintegration of a cell. It is interesting that two slightly different caspase substrates revealed distinct localization: DEVD–G1 (Oncoimmunin) showed staining only in fragments with nuclei, while FITC–VAD–FMK (Promega) localized to cytoplasmic fragments without DNA. These differences may be explained by preferential activity of different caspases responsible for cleavage of these slightly different substrates. This also may explain why these substrate inhibitors alone cannot inhibit cellular spontaneous fragmentation in mouse embryos (Xu et al., 2001), while they are effective in inhibiting cellular fragmentation of murine oocytes (Perez et al., 1997), since distinct triggers in different developmental stages may activate different caspases.
Despite the controversy of whether apoptosis is or is not responsible for induction of cellular fragmentation, it is becoming evident that embryo viability is associated with alterations in expression of cell death regulatory molecules. This is supported by recent reports on alteration of Bcl-2 and Bax protein ratios in bovine oocytes and embryos of variable quality (Yang and Rajamahendran, 2002) as well as elevated expression of Bcl-2 and Bax during compaction of human embryos (Spanos et al., 2002). Moreover, changes in expression of Fas, Fas-ligand and Bax mRNA in human viable and non-viable embryos also support this hypothesis (Liu et al., 2000; Kawamura et al., 2001). While these reports only assess the transcript expression for these cell death-associated molecules, increased accumulation of Bax (Antczak and Van Blerkom, 1999) and Hrk protein (current report) as well as elevated caspase-like activity (Martinez et al., 2002) in cellular fragments and some blastomeres suggest that interaction or balance between various cell death regulatory gene products within subcellular domains may function as a primary determinant regulating cellular fragmentation during mammalian preimplantation embryo development.
While the overall trend of expression for some genes among embryos remained unchanged between experiments, others showed variable levels. We attribute this discrepancy in the mRNA levels to an inherent variability of gene expression by human oocytes and embryos as was previously reported (Holding et al., 2000; Steuerwald et al., 2000). However, other alternative explanations are also possible. First, the entire preimplantation embryo was processed for mRNA analysis. Consequently, it is possible that comparable analyses of individual blastomeres prior to and after the initiation of fragmentation would yield quantitatively different results. Second, fragmented blastomeres degrade their nuclear and cytoplasmic compartments. Therefore, depending upon the degree of fragmentation, not all blastomeres in any given embryo may contribute to the pool of mRNA used for analysis. Third, PCD is an active cellular process, and thus normal-appearing blastomeres (and embryos) may have already begun to alter their patterns of gene expression in preparation for cell death induction. Moreover, we cannot exclude the possibility that these studied proteins are regulated at the post-translational level, via protein modification, sequestration and complex–protein interaction as reported for many somatic cells (Adams and Cory, 1998). Thus, further functional studies using both human and animal models are required to resolve the relationship between regulation of cell death pathways and cellular fragmentation of preimplantation embryos. However, elevated expression of Hrk transcript and protein in a subset of fragmented embryos supports involvement of Bcl-2 family members in regulation of embryo survival.
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Acknowledgements |
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We would like to thank the following scientists for providing cDNA or antibody used in this study: Dr S.Cory for Bcl-w; Dr G.Nunez for Hrk; Dr E.Alnemri for Caspase-3; and Dr S.Shin for Bfl-1. We would also like to thank Dr G.I.Perez for technical assistance with the single cell caspase activity assay and to Dr T.Matikainen for helpful suggestions during the preparation of this manuscript. This study was supported by a grant from CIHR.
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