Caspase Activity in Preimplantation Human Embryos Is Not Associated with Apoptosis

Human Reproduction Vol.17, No.6 pp. 1584–1590, 2002

Caspase activity in preimplantation human embryos is not associated with apoptosis

Francisco Martinez1, Laura Rienzi2, Marcello Iacobelli2, Filippo Ubaldi2, Carmen Mendoza1,3, Ermanno Greco2 and Jan Tesarik3,4

1University of Granada, Campus Universitario Fuentenueva, Granada, Spain, 2European Hospital, Rome, Italy and 3MAR&Gen, Molecular Assisted Reproduction and Genetics, Granada, Spain 4To whom correspondence should be addressed at: MAR&Gen, Molecular Assisted Reproduction and Genetics, Gracia 36, 18002 Granada, Spain. E-mail: [email protected]

BACKGROUND: Previous studies on mammalian preimplantation embryos have suggested an association between caspase activation, blastomere fragmentation and apoptosis. However, some reports on human embryos questioned the causal relationship between blastomere fragmentation and apoptosis, and information about the presence and activity of caspases in human embryos is lacking. METHODS: A fluorochrome-labelled universal caspase inhibitor was used to visualize active caspases in blastomeres and fragments of preimplantation human embryos. RESULTS: Caspase activity was detected only after fertilization, and was rare in blastomeres but frequent in fragments. The incidence of caspase activity in blastomeres and fragments was stable between the 2-cell and 12-cell stages. Caspase- positive blastomeres were only seen in poor-morphology embryos. The percentage of caspase-positive fragments was increased in embryos with multinucleated blastomeres but was unrelated to embryo morphology. Moreover, caspase-positive fragments detached from healthy blastomeres that were isolated by embryo biopsy and subsequently underwent mitotic division in culture. CONCLUSIONS: These data suggest that caspases in preimplantation human embryos are involved in developmental processes unrelated to cell death.

Key words: apoptosis/blastomere multinucleation/caspase/ploidy/preimplantation embryo

Introduction circumstances. However, the methodology (Levy et al., 1998) The preimplantation period of human embryonic development and interpretation of the results (Jurisicova et al., 1996) of is marked by a high incidence of cell death. This phenomenon those studies have been challenged (Van Blerkom et al., may concern only a minority of blastomeres, allowing survival 2001). The exact nature of these changes thus remains to be of the whole embryo, or it may involve most or all of determined. Moreover, some studies failed to find signs of the blastomeres and lead to embryo demise. Cell death in apoptosis in preimplantation human embryos (Hardy, 1999; preimplantation embryos is often associated with fragmenta- Van Blerkom et al., 2001). If the above phenomena are indeed tion, and several studies have demonstrated a relationship due to apoptosis, they should be accompanied by the activation between the number and volume of fragments, on the one hand, of specific regulatory proteins involved in this process in other and developmental competence of cleavage stage embryos on cell types. the other hand (Giorgetti et al., 1995; Hoover et al., 1995; This study was undertaken to evaluate the activity of a Alikani et al., 1999; Antczak and Van Blerkom, 1999; Gerris family of cytoplasmic cysteine proteases which are involved et al., 1999). Because fragmentation is a typical morphological in both the signalling and the execution phase of apoptosis consequence of programmed cell death (apoptosis) in a variety (Thornberry and Lazebnik, 1998). Active caspases were visual- of other cell types, it has been suggested that the appearance ized in living embryo blastomeres and cell fragments with the of fragments in preimplantation embryos is associated with use of a fluorescein-tagged universal caspase inhibitor which the activation of apoptosis which may cause the loss of binds active caspases, but not their inactive zymogen forms. individual blastomeres or the death of the whole embryo The presence and distribution of active caspases were related (Jurisicova et al., 1996; Levy et al., 1998). to the stage of preimplantation embryo development, embryo Previous studies on apoptosis of human embryos (Jurisicova morphology, ploidy and the presence of multinucleated blas- et al., 1996; Levy et al., 1998) used outcome measures that tomeres. The working hypothesis to be tested was that cell detect consequences of the cell autodestruction process (plasma death in human preimplantation embryos leads to blastomere membrane phosphatidylserine externalization and DNA frag- fragmentation accompanied by caspase activation. Accord- mentation) which may also result from necrosis in some ingly, the following outcomes were predicted: (i) the presence

1584 © European Society of Human Reproduction and Embryology Caspase activity in embryos of caspase activity in fragments and degenerating blastomeres; (four at the 2-cell stage, two at the 3-cell stage, three at the 4-cell (ii) the absence of caspase activity in dividing blastomeres; stage, one at the 5-cell stage, and two at the 6-cell stage) by and (iii) a relationship between the presence of active caspases using terminal deoxynucleotidyltransferase-mediated dUTP nick-end and embryo morphology. The data obtained were only partly labelling (TUNEL) with the Cell Death Detection Kit (Boehringer, consistent with these predicted outcomes, and suggested that Mannheim, Germany) containing fluoroscein isothiocyanate (FITC)- labelled dUTP (detection of fragmented DNA) and propidium iodide caspases in human preimplantation embryos are involved in (detection of total DNA). After the removal of the zona pellucida by apoptosis-unrelated events. a brief incubation (37°C, 20 s) with 0.5% pronase (Sigma, St Louis, Missouri, USA), embryos were fixed with 5% glutaraldehyde in Materials and methods 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 Source and classification of oocytes and embryos incubated in Cell Death Detection Kit reagents according to the This study involved nine unfertilized metaphase II oocytes and 55 manufacturer’s instructions. After mounting in Slow-Fade lite embryos of which nine were at the pronuclear stage, 14 at the 2-cell (Molecular Probes, Eugene, OR, USA), the embryos were examined stage, 11 at the 3- or 4-cell stage, 12 at the 5- to 8-cell stage, and alternatively by epifluorescence and phase-contrast microscopy. nine at the 10- to 12-cell stage. An additional 12 embryos (four at the 2-cell stage, five at the 3- or 4-cell stage, and three at the 5- or Visualization of active caspases in intact embryos 6-cell stage) were used in a preliminary study aimed at the detection Active caspases were visualized in living embryos with the use of of intact and fragmented DNA in blastomeres and fragments. The CaspACE™ FITC-VAD-FMK in-situ marker (Promega, Madison, unfertilized oocytes were those that failed to fertilize after conventional WI, USA). FITC-VAD-FMK is a fluorescent analogue of the cell IVF (n ϭ 2) or ICSI (n ϭ 7); these were allocated to this study at permeable pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl- 24–32 h after in-vitro insemination or ICSI. The embryos were either analyl-aspartyl-[O-methyl]-fluoromethylketone) in which the FITC those resulting from abnormal fertilization (one pronucleus and group has been substituted for the carboxybenzoyl (Z) group to create two polar bodies, indicative of parthenogenetic activation, or three the fluorescent marker that penetrates into intact living cells where it pronuclei and one polar body, indicative of gynogenetic triploidy) or binds irreversibly to activated caspases. FITC-VAD-FMK was dis- those resulting from normal fertilization (two pronuclei and two solved in IVF-20 medium (Scandinavian IVF Science, Gothenborg, polar bodies) with development arrested spontaneously during post- Sweden) to a concentration of 250 µmol/l. This stock solution was fertilization in-vitro culture. In the former case, embryos were divided into aliquots and kept frozen at –20°C until use. The final allocated to this study after the detection of fertilization anomaly, incubation medium was prepared shortly before each incubation by and were then either processed immediately for active caspase diluting the stock solution of FITC-VAD-FMK with IVF-20 medium visualization or allowed to undergo one or several cleavage divisions to a concentration of 10 µmol/l. Embryos were incubated in this during subsequent in-vitro culture before processing. In the latter solution at 37°C under a gas phase of 5% CO in air for 12 h. After case, embryos were allocated to this study only when it was clear 2 incubation, embryos were washed twice in IVF medium and examined that their development had been arrested irreversibly; this conclusion immediately by combined phase-contrast and fluorescence was made when the time the embryos had spent in culture was at microscopy. least 24 h longer than the normal cleavage timing (at least 2-cell To exclude the possibility of non-specific fluorescence caused by stage 2 days after fertilization, at least 4-cell stage 3 days after plasma membrane damage, 10 embryos previously stained with FITC- fertilization, at least 8-cell stage 4 days after fertilization, and at least VAD-FMK and examined in the living state were subsequently 16-cell stage 5 days after fertilization) and when no cell of the exposed for 30 min to hypotonic conditions (0.5% sodium citrate) embryo had undergone a cleavage division during the last 24 h and processed with FITC-VAD-FMK again. No difference in the in culture. number of positively staining blastomeres or fragments was detected Morphology of cleaving embryos was evaluated as described in the same embryos before and after the membrane-destabilising (Tesarik and Greco, 1999). Accordingly, embryos with equal-sized treatment. blastomeres, with Ͻ10% of intrazonal space occupied by fragments and with clear, non-granulated cytoplasm in all blastomeres Visualization of active caspases in isolated blastomeres (Figure 1A) were referred to as good-morphology embryos throughout this study. Embryos that failed to fulfil at least one of these criteria Blastomeres were isolated from embryos with the use of Cook (Figure 1B) were referred to as poor-morphology embryos. The only blastomere aspiration pipette (Type UCL/Hammersmith; internal µ exception to the application of the above criteria was the 3-cell stage diameter, 35 m; Cook Australia, Queensland, Australia) which was at which embryos with one bigger and two smaller, equal-sized inserted by means of Narishige micromanipulators (Narishige, Tokyo, blastomeres were considered as good-morphology embryos, whereas Japan) through a hole in the zona pellucida previously opened by those with three equal-sized blastomeres considered as poor- using a non-contact surgical laser equipment (Fertilase; Medical morphology embryos. Technologies Montreux, Switzerland). After 24 h of culture, blastom- Between the 2-cell and 6-cell stages, embryos were also examined eres, together with eventually present newly formed fragments arising for the presence of multinucleated blastomeres (MNB). An embryo during in-vitro culture after blastomere isolation, were treated with was considered as having MNB when at least one multinucleated FITC-VAD-FMK and examined as described in the previous section, blastomere was detected at these early cleavage stages, even when except for the time of incubation which was reduced to 1 h. no more MNB were seen at later cleavage stages when the embryo Quantitative analysis was processed for active caspase visualization. During examination by fluorescence microscopy, cells and fragments Detection of intact and fragmented DNA in blastomeres and binding FITC-VAD-FMK were counted in each embryo. Structures fragments were considered as cells when they were Ͼ20 µm in diameter and The evaluation of the presence of intact and fragmented DNA in when a nucleus was detected in them by phase-contrast microscopy. blastomeres and cell fragments was performed on 12 cleaving embryos Structures measuring ഛ20 µm in diameter were considered as 1585 F.Martinez et al.

Figure 1. Examples of good-morphology (A) and poor-morphology (B) embryos. Scale bar ϭ 50 µm. fragments. Structures measuring Ͼ20 µm but lacking a detectable nucleus were excluded from the quantitative analysis. The overall Table I. Incidence of caspase activity in embryo cells and fragments at number of cells and fragments was also determined after switching different stages of preimplantation development to the phase-contrast mode of microscopic observation. The proportion of caspase-positive cells and fragments was then calculated and Stage No. of Caspase activity specimens analysed in relation to embryo developmental stage, morphology, Proportion (%) of Proportion (%) of ploidy and the presence or absence of MNB. The quantitative analysis cells fragments was restricted to embryos with less than nine cells because the distinction between cells and fragments becomes increasingly difficult Pronucleate zygote 9 0/9 (0)a 1/4 (25)a at more advanced stages of preimplantation development. 2-cell 14 0/28 (0)a 17/33 (52)a 3- and 4-cell 11 1/42 (2)a 22/41 (54)a Statistical analysis 5- to 8-cell 12 2/76 (3)a 26/46 (57)a Percentages of blastomeres and cell fragments containing caspase a Ͼ activity were compared in individual categories of embryos using the Differences between individual stages were not significant (P 0.05). χ2-test. themselves and the corresponding first polar bodies. After fertilization, caspase activity was never observed in pronucle- Results ated zygotes and was rare in blastomeres of cleaving embryos In a preliminary study involving 12 embryos between the (Table I). On the other hand, caspase activity was frequently 2- and 6-cell stages, intact—but not fragmented—DNA was detected in fragments observed at individual cleavage stages, detected in all structures considered to be cells based on the and the differences between individual stages were not signi- previous size evaluation (Ͼ20 µm in diameter). Intact DNA ficant (Table I). Caspase activity was also present in a single was detected in only two out of 48 fragments (4.2%) observed structure of Ͻ20 µm in diameter in one pronucleate zygote. in these embryos. This finding concerned one embryo at the These structures (Ͻ20 µm) appeared in each of the three 2-cell stage and one embryo at the 3-cell stage, both of which zygotes at the pronuclear stage (one with two pronuclei and showed the presence of MNB. Both of these fragments were two with one pronucleus), and was probably the second polar TUNEL-negative. Based on this pilot study, the size limit of body. In one of these three zygotes, another caspase-positive 20 µm was applied in order to distinguish embryonic cells and structure, considered to be a fragment, was observed (Table I). fragments throughout this study. Caspase activity was evaluated The remaining six zygotes at the pronuclear stage showed in nine unfertilized metaphase II oocytes and in 55 preimplanta- three pronuclei, and did not extrude the second polar body. tion embryos ranging between the pronuclear zygote stage and No similar caspase-positive structure was observed in these the 12-cell stage. In addition, caspase activity was investigated zygotes. in six blastomeres isolated from four multifragmented embryos. The incidence of caspase activity among embryo blastomeres Relationship between caspase activity and embryo mor- and fragments was related to the developmental stage, embryo phology morphology and the presence of MNB. Among 37 cleaving embryos, caspase-positive blastomeres were detected in only three embryos. In all these cases, only Relationship between caspase activity and embryo develop- one blastomere per embryo showed caspase activity (Figure mental stage 2A and 2B). Consequently, only three blastomeres out of 146 No caspase activity was detected in any of the nine unfertilized evaluated were caspase-positive, and all of these belonged to oocytes involved in this study. This applied to both the oocytes the embryo group scored as poor-morphology (Table II). 1586 Caspase activity in embryos

Table II. Incidence of caspase activity in embryo cells and fragments as related to cleaving embryo morphology

Morphology No. of Caspase activity specimens Proportion (%) of Proportion (%) of cells fragments

Good 14 0/51 (0)a 18/44 (41)a Poor 18 3/95 (3)a 71/157 (45)a

aDifferences between individual stages were not significant (P Ͼ 0.05).

Table III. Incidence of caspase activity in embryo cells and fragments as related to embryo ploidy and the presence of multinucleated blastomeres (MNB)a

Embryos No. of Caspase activity specimens Proportion (%) Proportion (%) of cells of fragments

Diploid without MNB 4 0/17 (0) 6/14 (43) Diploid with MNB 2 0/8 (0) 7/8 (88) Triploid without MNB 9 0/68 (0) 22/49 (45)b Triploid with MNB 8 1/47 (2) 38/47 (81)c

aThis analysis was limited to embryos between the 2-cell and 6-cell stages, because of difficulties in visualizing MNB at later stages, and did not include embryos resulting from unipronucleated zygotes because of uncertainty about their final ploidy. Diploid embryos are those developing from zygotes showing two pronuclei and two polar bodies; triploid embryos are those developing from zygotes showing three pronuclei and only one polar body. b,cValues with different superscript letters were significantly different (P Ͻ 0.05).

In contrast to blastomeres, the incidence of caspase activity was much higher in fragments observed both in good-morpho- logy (Figure 2C and 2D) and poor-morphology (Figure 2E and 2F) embryos. However, the proportion of caspase-positive fragments did not differ between the good-morphology and poor-morphology embryos (Table II).

Relationship between caspase activity and blastomere multin- ucleation In this comparison, cleaving embryos developing from diploid Figure 2. Phase-contrast (A, C, E, G) and fluorescence (B, D, F, and triploid zygotes were involved. An embryo was considered H) micrographs of embryos and isolated blastomeres treated with as one with MNB when at least one multinucleated blastomere FITC-VAD-FMK to detect caspase activity. (A, B) Poor- was detected during one of the repeated microscopic examina- morphology 8-cell embryo showing caspase activity in one blastomere (arrow) in addition to several caspase-positive tions between the 2-cell and 6-cell stages. If no such blastomere fragments. Scale bar ϭ 50 µm. (C, D) Good-morphology 10-cell was found during these examinations, an embryo was classified embryo showing caspase activity in numerous small and dispersed as being without MNB. This classification was applied irre- cell fragments (some of which are out of focus). Scale bar ϭ spective of whether MNB could be seen at the time of caspase µ 50 m. (E, F) Poor-morphology 4-cell embryo showing caspase visualization. activity in locally accumulated fragments but not in a persisting healthy-appearing blastomere. Scale bar ϭ 50 µm. (G, H) Pair of Caspase activity was only detected in one blastomere of a blastomeres resulting from mitotic division of an apparently healthy triploid embryo with MNB (Table III). The blastomere in blastomere isolated from a multifragmented embryo. The question had four nuclei. However, the incidence of caspase blastomere was devoid of fragments at the time of isolation. activity in fragments was higher in embryos with MNB, Caspase activity is present in one of the fragments (arrow) that irrespective of whether they originated from diploid or triploid detached from the isolated blastomere during the culture period. Scale bar ϭ 30 µm. zygotes (Table III) and reached statistical significance for triploid embryos. 1587 F.Martinez et al.

Study of isolated blastomeres yet failed to respond by triggering the activation reaction. In order to address the dynamics of caspase activation, six Interestingly, in previous studies fragmented DNA was detected normal-appearing mononucleated blastomeres were isolated in the first polar body of unfertilized mouse (Fujino et al., from four multifragmented embryos, freed from any attached 1996) and human oocytes (Van Blerkom and Davis, 1998), cell fragments and cultured under standard embryo culture leading to the suggestion that first polar body elimination conditions (Rienzi et al., 1998) for an additional 24 h to occurs by apoptosis. The present findings tend to suggest that, allow further cleavage division. Among 11 mononucleated if apoptosis is involved in polar body DNA fragmentation, it blastomeres isolated from eight highly fragmented embryos, may proceed by a caspase-independent mechanism. A recent seven blastomeres underwent further mitotic division during study has demonstrated that apoptotic DNA fragmentation the subsequent 24 h of culture. No fragments were left in patterns can occur in the absence of the caspase pathway, by association with the isolated blastomeres at the beginning of means of endonuclease G release from mitochondria and culture. Notwithstanding, new fragments were detached during translocation to the nucleus, which bypasses the cytoplasmic the culture from each of the four blastomeres that underwent apoptotic cascade (Li et al., 2001) For the polar body, this mitotic division. Caspase activity was absent from all isolated could result in positive TUNEL fluorescence—as has been blastomeres at the end of culture, but was present in some of reported by several groups—in the absence of detectable the newly formed fragments extruded from the four divided caspase activity. blastomeres (Figure 2G and 2H). 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), Discussion is another important milestone in preimplantation embryo Previous studies (Jurisicova et al., 1996; Levy et al., 1998) development. In Xenopus embryos, the activation of embryonic have led to the hypothesis that activation of apoptotic pathways gene transcription leads to a decrease in embryo susceptibility leads to blastomere fragmentation in the preimplantation to apoptosis after the previous increase due to oocyte activation embryo. In order to investigate the correlation between caspase at fertilization (Sible et al., 1997). In mouse embryos, the activation and blastomere fragmentation, caspase activity was appearance of mRNA for several types of caspases was examined in arrested human embryonic blastomeres and frag- reported to coincide with the onset of embryonic gene transcrip- ments. It was found that caspase activity in preimplantation tion (Exley et al., 1999), although the design of that study human embryos is not correlated either with apoptosis or with does not exclude the possibility that some of the structures blastomere fragmentation. Although caution must be shown considered to be fragmented embryos were in fact fragmented when comparing results obtained from arrested human embryos unfertilized oocytes. In the present study, no difference in the with those obtained from developmentally competent embryos proportion of caspase-positive fragments was observed between of other species, both types of study support the novel developmental stages before and after embryonic gene hypothesis that caspase activation can occur via a non-apoptotic activation. mechanism and may have a formative, rather than a destruc- Interestingly, caspase activity was only rarely seen in embryo tive, role. blastomeres as compared with cell fragments. Moreover, all Caspase expression has previously been studied in mouse fragments observed in cleaving embryos certainly did not (Jurisicova et al., 1998; Moley et al., 1998; Exley et al., 1999) arise from complete fragmentation of a blastomere because and rat (Hinck et al., 2001) preimplantation embryos. However, fragments—both with and without caspase activity—were also only one of these studies (Exley et al., 1999) compared observed in good-morphology embryos which apparently had caspase expression levels at different stages of preimplantation not lost a blastomere. The possibility that fragments within development. mRNA for most of the caspases studied was preimplantation embryos may arise by mechanisms other than present in unfertilized oocytes, undetectable in zygotes and complete blastomere disintegration is corroborated by the present again from the 2-cell stage onwards (Exley et al., 1999). observation that the incidence of cytoplasmic fragmentation However, caspase protein must have been present in both in mouse embryos is not affected by caspase inhibitors (Xu zygotes and cleaving embryos because caspase activity was et al., 2001) and by the finding that the fragments developing detected in the second polar bodies, staurosporine-treated in human embryos can be transient structures some of which are zygotes and fragmented embryos by using a selective group- actually ‘pseudo-fragments’ retaining cytoplasmic continuity II caspase substrate (Exley et al., 1999). Similarly, in the with the underlying blastomere (Van Blerkom et al., 2001). present study caspase activity was found in the second polar The caspase pathway may indeed be part of the apoptotic bodies, in fragments attached to zygotes, and in blastomeres process in some fragments, and the observed differences and fragments of cleaving embryos, but not in unfertilized between fragments in caspase activity may be related to oocytes or in the first polar bodies. This was the first demonstra- fragment size and mitochondrial number. Apparent differences tion that, in spite of the probable presence of caspases in in mitochondrial numbers between fragments have been unfertilized oocytes, caspase activation does not occur until reported by electron microscopy studies (Jurisicova et al., oocyte activation by the fertilizing spermatozoon. The simple 1996; Van Blerkom et al., 2001). It is possible that in presence of a spermatozoon within the oocyte is not sufficient comparatively large fragments with few mitochondria, or in for this process, because most of the unfertilized oocytes used smaller fragments with proportionally larger mitochondrial in this study had a spermatozoon injected into their cytoplasm numbers, mitochondrial deterioration occurs over relatively 1588 Caspase activity in embryos brief periods of time and results in cytochrome c release, involved in yet unknown processes during which fragments which would convert the procaspase enzyme(s) to an active temporarily or durably detach from healthy blastomeres, and or mature form. In this sense, caspase activity would serve to that the activation of caspases within fragments is enhanced in destabilize the fragments using a portion of the apoptotic embryos with MNB. Thus, caspases in human preimplantation cascade in a novel manner. embryos may have a double function: (i) a destructive one, Although the present study did not reveal any relationship serving to free the embryo from unnecessary, irreparably between the embryo cleavage stage and morphology on the damaged or potentially harmful cellular components; and (ii) one hand, and the proportion of caspase-positive fragments on a formative one, helping the embryo to shape blastomeres to the other hand, the proportional increase in caspase-positive developmentally regulated structural and functional patterns. fragments was associated with blastomere multinucleation in On the other hand, these data fail to support the activity of both the diploid and triploid embryos. 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