1 University of Granada, Campus Universitario Fuentenueva, Granada, Spain, 2 European Hospital, Rome, Italy and 3 MAR&Gen, Molecular Assisted Reproduction and Genetics, Granada, Spain
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Abstract |
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Key words: apoptosis/blastomere multinucleation/caspase/ploidy/preimplantation embryo
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Introduction |
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Previous studies on apoptosis of human embryos (Jurisicova et al., 1996; Levy et al., 1998
) used outcome measures that detect consequences of the cell autodestruction process (plasma membrane phosphatidylserine externalization and DNA fragmentation) which may also result from necrosis in some circumstances. However, the methodology (Levy et al., 1998
) and interpretation of the results (Jurisicova et al., 1996
) of those studies have been challenged (Van Blerkom et al., 2001
). The exact nature of these changes thus remains to be determined. Moreover, some studies failed to find signs of apoptosis in preimplantation human embryos (Hardy, 1999
; Van Blerkom et al., 2001
). If the above phenomena are indeed due to apoptosis, they should be accompanied by the activation of specific regulatory proteins involved in this process in other cell types.
This study was undertaken to evaluate the activity of a family of cytoplasmic cysteine proteases which are involved in both the signalling and the execution phase of apoptosis (Thornberry and Lazebnik, 1998). Active caspases were visualized in living embryo blastomeres and cell fragments with the use of a fluorescein-tagged universal caspase inhibitor which binds active caspases, but not their inactive zymogen forms. The presence and distribution of active caspases were related to the stage of preimplantation embryo development, embryo morphology, ploidy and the presence of multinucleated blastomeres. The working hypothesis to be tested was that cell death in human preimplantation embryos leads to blastomere fragmentation accompanied by caspase activation. Accordingly, the following outcomes were predicted: (i) the presence of caspase activity in fragments and degenerating blastomeres; (ii) the absence of caspase activity in dividing blastomeres; and (iii) a relationship between the presence of active caspases and embryo morphology. The data obtained were only partly consistent with these predicted outcomes, and suggested that caspases in human preimplantation embryos are involved in apoptosis-unrelated events.
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Materials and methods |
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Morphology of cleaving embryos was evaluated as described (Tesarik and Greco, 1999). Accordingly, embryos with equal-sized blastomeres, with <10% of intrazonal space occupied by fragments and with clear, non-granulated cytoplasm in all blastomeres (Figure 1A
) were referred to as good-morphology embryos throughout this study. Embryos that failed to fulfil at least one of these criteria (Figure 1B
) were referred to as poor-morphology embryos. The only exception to the application of the above criteria was the 3-cell stage at which embryos with one bigger and two smaller, equal-sized blastomeres were considered as good-morphology embryos, whereas those with three equal-sized blastomeres considered as poormorphology embryos.
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Detection of intact and fragmented DNA in blastomeres and fragments
The evaluation of the presence of intact and fragmented DNA in blastomeres and cell fragments was performed on 12 cleaving embryos (four at the 2-cell stage, two at the 3-cell stage, three at the 4-cell stage, one at the 5-cell stage, and two at the 6-cell stage) by using terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) with the Cell Death Detection Kit (Boehringer, Mannheim, Germany) containing fluoroscein isothiocyanate (FITC)-labelled dUTP (detection of fragmented DNA) and propidium iodide (detection of total DNA). After the removal of the zona pellucida by a brief incubation (37°C, 20 s) with 0.5% pronase (Sigma, St Louis, Missouri, USA), embryos were fixed with 5% glutaraldehyde in 0.05 mol/l cacodylate buffer (pH 7.4) for 1 h and washed in 0.1 mol/l cacodylate buffer overnight. Zona-free embryos were then incubated in Cell Death Detection Kit reagents according to the manufacturer's instructions. After mounting in Slow-Fade lite (Molecular Probes, Eugene, OR, USA), the embryos were examined alternatively by epifluorescence and phase-contrast microscopy.
Visualization of active caspases in intact embryos
Active caspases were visualized in living embryos with the use of CaspACETM FITC-VAD-FMK in-situ marker (Promega, Madison, WI, USA). FITC-VAD-FMK is a fluorescent analogue of the cell permeable pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-analyl-aspartyl-[O-methyl]-fluoromethylketone) in which the FITC group has been substituted for the carboxybenzoyl (Z) group to create the fluorescent marker that penetrates into intact living cells where it binds irreversibly to activated caspases. FITC-VAD-FMK was dissolved in IVF-20 medium (Scandinavian IVF Science, Gothenborg, Sweden) to a concentration of 250 µmol/l. This stock solution was divided into aliquots and kept frozen at 20°C until use. The final incubation medium was prepared shortly before each incubation by diluting the stock solution of FITC-VAD-FMK with IVF-20 medium to a concentration of 10 µmol/l. Embryos were incubated in this solution at 37°C under a gas phase of 5% CO2 in air for 12 h. After incubation, embryos were washed twice in IVF medium and examined immediately by combined phase-contrast and fluorescence microscopy.
To exclude the possibility of non-specific fluorescence caused by plasma membrane damage, 10 embryos previously stained with FITC-VAD-FMK and examined in the living state were subsequently exposed for 30 min to hypotonic conditions (0.5% sodium citrate) and processed with FITC-VAD-FMK again. No difference in the number of positively staining blastomeres or fragments was detected in the same embryos before and after the membrane-destabilising treatment.
Visualization of active caspases in isolated blastomeres
Blastomeres were isolated from embryos with the use of Cook blastomere aspiration pipette (Type UCL/Hammersmith; internal diameter, 35 µm; Cook Australia, Queensland, Australia) which was inserted by means of Narishige micromanipulators (Narishige, Tokyo, Japan) through a hole in the zona pellucida previously opened by using a non-contact surgical laser equipment (Fertilase; Medical Technologies Montreux, Switzerland). After 24 h of culture, blastomeres, together with eventually present newly formed fragments arising during in-vitro culture after blastomere isolation, were treated with FITC-VAD-FMK and examined as described in the previous section, except for the time of incubation which was reduced to 1 h.
Quantitative analysis
During examination by fluorescence microscopy, cells and fragments binding FITC-VAD-FMK were counted in each embryo. Structures were considered as cells when they were >20 µm in diameter and when a nucleus was detected in them by phase-contrast microscopy. Structures measuring 20 µm in diameter were considered as fragments. Structures measuring >20 µm but lacking a detectable nucleus were excluded from the quantitative analysis. The overall number of cells and fragments was also determined after switching to the phase-contrast mode of microscopic observation. The proportion of caspase-positive cells and fragments was then calculated and analysed in relation to embryo developmental stage, morphology, ploidy and the presence or absence of MNB. The quantitative analysis was restricted to embryos with less than nine cells because the distinction between cells and fragments becomes increasingly difficult at more advanced stages of preimplantation development.
Statistical analysis
Percentages of blastomeres and cell fragments containing caspase activity were compared in individual categories of embryos using the 2-test.
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Results |
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Relationship between caspase activity and embryo developmental stage
No caspase activity was detected in any of the nine unfertilized oocytes involved in this study. This applied to both the oocytes themselves and the corresponding first polar bodies. After fertilization, caspase activity was never observed in pronucleated zygotes and was rare in blastomeres of cleaving embryos (Table I). On the other hand, caspase activity was frequently detected in fragments observed at individual cleavage stages, and the differences between individual stages were not significant (Table I
). Caspase activity was also present in a single structure of <20 µm in diameter in one pronucleate zygote. These structures (<20 µm) appeared in each of the three zygotes at the pronuclear stage (one with two pronuclei and two with one pronucleus), and was probably the second polar body. In one of these three zygotes, another caspase-positive structure, considered to be a fragment, was observed (Table I
). The remaining six zygotes at the pronuclear stage showed three pronuclei, and did not extrude the second polar body. No similar caspase-positive structure was observed in these zygotes.
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Relationship between caspase activity and blastomere multinucleation
In this comparison, cleaving embryos developing from diploid and triploid zygotes were involved. An embryo was considered as one with MNB when at least one multinucleated blastomere was detected during one of the repeated microscopic examinations between the 2-cell and 6-cell stages. If no such blastomere was found during these examinations, an embryo was classified as being without MNB. This classification was applied irrespective of whether MNB could be seen at the time of caspase visualization.
Caspase activity was only detected in one blastomere of a triploid embryo with MNB (Table III). The blastomere in question had four nuclei. However, the incidence of caspase activity in fragments was higher in embryos with MNB, irrespective of whether they originated from diploid or triploid zygotes (Table III
) and reached statistical significance for triploid embryos.
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Discussion |
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Caspase expression has previously been studied in mouse (Jurisicova et al., 1998; Moley et al., 1998
; Exley et al., 1999
) and rat (Hinck et al., 2001
) preimplantation embryos. However, only one of these studies (Exley et al., 1999
) compared caspase expression levels at different stages of preimplantation development. mRNA for most of the caspases studied was present in unfertilized oocytes, undetectable in zygotes and present again from the 2-cell stage onwards (Exley et al., 1999
).
However, caspase protein must have been present in both zygotes and cleaving embryos because caspase activity was detected in the second polar bodies, staurosporine-treated zygotes and fragmented embryos by using a selective group-II caspase substrate (Exley et al., 1999). Similarly, in the present study caspase activity was found in the second polar bodies, in fragments attached to zygotes, and in blastomeres and fragments of cleaving embryos, but not in unfertilized oocytes or in the first polar bodies. This was the first demonstration that, in spite of the probable presence of caspases in unfertilized oocytes, caspase activation does not occur until oocyte activation by the fertilizing spermatozoon. The simple presence of a spermatozoon within the oocyte is not sufficient for this process, because most of the unfertilized oocytes used in this study had a spermatozoon injected into their cytoplasm yet failed to respond by triggering the activation reaction. Interestingly, in previous studies fragmented DNA was detected in the first polar body of unfertilized mouse (Fujino et al., 1996
) and human oocytes (Van Blerkom and Davis, 1998
), leading to the suggestion that first polar body elimination occurs by apoptosis. The present findings tend to suggest that, if apoptosis is involved in polar body DNA fragmentation, it may proceed by a caspase-independent mechanism. A recent study has demonstrated that apoptotic DNA fragmentation patterns can occur in the absence of the caspase pathway, by means of endonuclease G release from mitochondria and translocation to the nucleus, which bypasses the cytoplasmic apoptotic cascade (Li et al., 2001
) For the polar body, this could result in positive TUNEL fluorescenceas has been reported by several groupsin the absence of detectable caspase activity.
After oocyte activation, embryonic genome activation, occurring between the 4-cell and 8-cell stages in human embryos (Tesarik et al., 1986, 1988
; Braude et al., 1988
), is another important milestone in preimplantation embryo development. In Xenopus embryos, the activation of embryonic gene transcription leads to a decrease in embryo susceptibility to apoptosis after the previous increase due to oocyte activation at fertilization (Sible et al., 1997
). In mouse embryos, the appearance of mRNA for several types of caspases was reported to coincide with the onset of embryonic gene transcription (Exley et al., 1999
), although the design of that study does not exclude the possibility that some of the structures considered to be fragmented embryos were in fact fragmented unfertilized oocytes. In the present study, no difference in the proportion of caspase-positive fragments was observed between developmental stages before and after embryonic gene activation.
Interestingly, caspase activity was only rarely seen in embryo blastomeres as compared with cell fragments. Moreover, all fragments observed in cleaving embryos certainly did not arise from complete fragmentation of a blastomere because fragmentsboth with and without caspase activitywere also observed in good-morphology embryos which apparently had not lost a blastomere. The possibility that fragments within preimplantation embryos may arise by mechanisms other than complete blastomere disintegration is corroborated by the observation that the incidence of cytoplasmic fragmentation in mouse embryos is not affected by caspase inhibitors (Xu et al., 2001) and by the finding that the fragments developing in human embryos can be transient structures some of which are actually `pseudo-fragments' retaining cytoplasmic continuity with the underlying blastomere (Van Blerkom et al., 2001
).
The caspase pathway may indeed be part of the apoptotic process in some fragments, and the observed differences between fragments in caspase activity may be related to fragment size and mitochondrial number. Apparent differences in mitochondrial numbers between fragments have been reported by electron microscopy studies (Jurisicova et al., 1996; Van Blerkom et al., 2001
). It is possible that in comparatively large fragments with few mitochondria, or in smaller fragments with proportionally larger mitochondrial numbers, mitochondrial deterioration occurs over relatively brief periods of time and results in cytochrome c release, which would convert the procaspase enzyme(s) to an active or mature form. In this sense, caspase activity would serve to destabilize the fragments using a portion of the apoptotic cascade in a novel manner.
Although the present study did not reveal any relationship between the embryo cleavage stage and morphology on the one hand, and the proportion of caspase-positive fragments on the other hand, the proportional increase in caspase-positive fragments was associated with blastomere multinucleation in both the diploid and triploid embryos. The difference between embryos with and without MNB was statistically significant only for triploid embryos, and not for diploid ones. This observation may be related to the process of pseudonucleus extrusion which frequently occurs in human MNB (Tesarik et al., 1987).
Previous studies dealing with the expression and activity of caspases in mammalian preimplantation embryos (Moley et al., 1998; Exley et al., 1999
; Hinck et al., 2001
; Xu et al., 2001
) only considered the death-inducing function of the caspase-mediated pathway. However, the experiments on isolated blastomeres described in the present study suggest that caspases may be activated in fragments detached from actively dividing, and thus apparently healthy, human embryonic cells. Caspases may thus have functions unrelated to cell death in preimplantation embryos. These findings also suggest that fragmentation occurs independently of apoptosis, and that caspase activation within fragments occurs secondarily and in only some fragments. In this context it might be pertinent to note that the concentration of staurosporine required to induce apoptosis in mouse preimplantation embryos (Exley et al., 1999
) was well above that commonly used in other cells and may thus have produced deleterious effects on the embryos which were unrelated to apoptosis induction.
The extrusion of caspase-positive fragments from supposedly healthy blastomeres may serve to sequester surplus cytoplasmic components which have become unnecessary in the actual context of the whole embryo's needs, and to isolate them from the bulk of cytoplasm before starting their dismantling by a topographically restricted apoptosis-like process. The polarization of a number of regulatory proteins in preimplantation human embryos (Antczak and Van Blerkom, 1999) may facilitate such a selective disposal. By analogy, several proteins known to be involved in apoptosis, including caspase-1, appear to participate in the formation of residual bodies in rat spermatids (Blanco-Rodriguez and Martinez-Garcia, 1999
), which also represents a physiological process unrelated to cell death. It remains to be determined whether caspases that become activated in some fragments remain active throughout the existence of the given fragment or whether they become inactive with time. If the latter is true, fragments may serve for temporal sequestration or destruction of unnecessary or harmful components. This role would be compatible with subsequent re-absorption of `burnt out' fragments by their mother cells.
In conclusion, the present study has shown that active caspases are rarely present in blastomeres, but are frequently present in fragments. These data suggest that caspases are involved in yet unknown processes during which fragments temporarily or durably detach from healthy blastomeres, and that the activation of caspases within fragments is enhanced in embryos with MNB. Thus, caspases in human preimplantation embryos may have a double function: (i) a destructive one, serving to free the embryo from unnecessary, irreparably damaged or potentially harmful cellular components; and (ii) a formative one, helping the embryo to shape blastomeres to developmentally regulated structural and functional patterns. On the other hand, these data fail to support the activity of the classical caspase signalling pathway in preimplantation human embryos and the implication of apoptosis in selective blastomere removal or blastomere fragmentation.
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Notes |
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References |
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Submitted on July 6, 2001; resubmitted on November 26, 2001; accepted on February 22, 2002.