REPRODUCTIONREVIEW

Focus on ART Deadly decisions: the role of genes regulating programmed cell death in human preimplantation embryo development

Andrea Jurisicova and Beth M Acton Division of Reproductive Sciences, Department of Obstetrics and Gynaecology and Department of Physiology, University of Toronto, Toronto, Ontario, Canada Correspondence should be addressed to A Jurisicova, Samuel Lunenfeld Research Institute Room 876, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5; Email: [email protected]

Abstract Human preimplantation embryo development is prone to high rates of early embryo wastage, particularly under current in vitro culture conditions. There are many possible underlying causes for embryo demise, including DNA damage, poor embryo metabolism and the effect of suboptimal culture media, all of which could result in an imbalance in gene expression and the failed execution of basic embryonic decisions. In view of the complex interactions involved in embryo development, a thorough understanding of these parameters is essential to improving embryo quality. An increasing body of evidence indi- cates that cell fate (i.e. survival/differentiation or death) is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins, many of which are expressed during oocyte and preimplantation embryo develop- ment. The recent availability of mutant mice lacking expression of various genes involved in the regulation of cell survival has enabled rapid progress towards identifying those molecules that are functionally important for normal oocyte and preimplan- tation embryo development. In this review we will discuss the current understanding of the regulation of cell death gene expression during preimplantation embryo development, with a focus on human embryology and a discussion of animal models where appropriate. Reproduction (2004) 128 281–291

Human in vitro fertilization and embryo wastage in mammalian preimplantation development have been studied extensively (Hardy 1999). One possible expla- Current data available from in vitro fertilization (IVF) prac- nation for apoptosis within the inner cell mass is that the tices in the USA indicate that there is a 27% live birth rate embryo is attempting to remove cells that have retained from all initiated cycles, which does show some improve- trophectodermal potential through selective killing via a ment over previous years (Jain et al. 2004). Embryo quality hydrogen peroxide mediated cell death pathway (Pierce remains a strong determining factor in the success rates of et al. 1989, 1991). However, the function of blastocyst IVF, as the live birth rate increases when there is a greater apoptosis remains unclear and will not be discussed selection of high quality embryos per cycle from which to further in this review. choose (Centers for Disease Control and Prevention In vitro fertilized embryos often display abnormal or 2003). One central difficulty in successful IVF therefore delayed cell division, frequently accompanied by cellular lies in the production of high quality embryos, as most fragmentation and developmental arrest before the blasto- embryos will succumb to embryo arrest, fragmentation, or cyst stage (Trounson & Bongso 1996) (Fig. 1). Cytoplasmic both. Even those embryos that are competent to reach the fragmentation is one of the most visible defects observed blastocyst stage in vitro are often subject to apoptosis in and is often used as an indicator of embryo quality. Studies both the inner cell mass and trophectodermal lineages. have demonstrated that pregnancy rates are inversely pro- The effects of cytokines on the rates of blastocyst growth portional to the degree of embryo fragmentation (Ebner and development have been studied extensively, yet the et al. 2001) and that the pattern of fragmentation plays a downstream effects on gene expression in the context of part in embryo quality (Alikani et al. 1999). Fragmented embryonic cell death remain relatively unexplored. A embryos typically contain variable sized blastomeres with description of the distinct cellular events known to be several cellular fragments that appear to maintain mem- associated with apoptosis occurring at the blastocyst stage brane integrity and exclude vital dyes (Antczak & Van

q 2004 Society for Reproduction and Fertility DOI: 10.1530/rep.1.00241 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/01/2021 09:50:56PM via free access 282 A Jurisicova and B M Acton

Figure 1 The fate of human and murine embryos. The top panel represents successful development from the zygote stage to the blastocyst stage. The timing of these events is different between species, human embryos taking a longer time to cleave to the blastocyst stage than murine embryos, but all embryos follow the same sequence of events. The middle panel represents two-cell arrest, the most frequently observed devel- opmental defect in murine embryos. After arrest, the two-cell embryo will degenerate, experience mild to moderate midline fragmentation, or fragment completely. The bottom panel represents human embryos, which often experience developmental difficulties between the four- and eight-cell stages. Embryos could then arrest, experience fragmentation coupled with the loss of blastomeres or fragmentation without the loss of blastomeres. Further discussion on the mechanisms and pathways involved in these modes of cell death can be found throughout the text.

Blerkom 1999). In most cases these anuclear cellular frag- tinue to fragment after treatment (A Jurisicova, unpublished ments will contain organelles and other cellular proteins observations). Thus it is possible that fragmentation is a (Jurisicova et al. 1996, Van Blerkom et al. 2002). The aver- normal coping mechanism of the embryo, and that it is the age blastomere size of fragmented embryos is significantly location or extent of embryo fragmentation that can nega- reduced by the degree of fragmentation (Hnida et al. tively influence developmental potential (Antczak & Van 2004), leading to decreased cellular content and thus Blerkom 1999). exerting a negative effect on the future mass of embryo. One mechanism that has been proposed to be the Embryo fragmentation, when accompanied by embryo cause of cytoplasmic fragmentation is the process of onco- cleavage and further development, is not by itself develop- sis (Van Blerkom et al. 2001). This is a form of cell death mentally lethal, as in some instances fragments were characterized by the formation of organelle-deprived cyto- resorbed or their presence did not impair embryo cleavage plasmic blebs that can either be resorbed by the cell or (Van Blerkom et al. 2001). Previously, it has been reported detach and float away under conditions of oxygen that fragment removal can improve the developmental deprivation (Majno & Joris 1995). As embryos are cultured potential of affected embryos (Alikani et al. 1999), as frag- in an oxygen-rich environment, it has been proposed that ments may restrict and compromise the process of com- similar morphological changes could occur in blastomeres paction. In our hands, fragment removal does not help that failed to inherit a mitochondrial complement capable embryos in which the fragments encompass more than of supporting its energy demands (Van Blerkom et al. 30% of the total embryo volume, as these embryos con- 2001). Mitochondrial membrane potential and energy

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Downloaded from Bioscientifica.com at 10/01/2021 09:50:56PM via free access PCD gene expression in human preimplantation embryos 283 production in preimplantation embryos have recently embryos from older IVF patients (Ziebe et al. 1997), and been the focus of much study and will be discussed by by an increased cell death index of blastocysts from older Van Blerkom in more detail elsewhere in this issue of female mice (Jurisicova et al. 1998b). Maternal factors that Reproduction. could contribute to abnormal preimplantation develop- Another hypothesis credits embryo fragmentation to the ment include cytoplasmic mRNAs, proteins, antioxidants, activation of programmed cell death pathways, especially small peptides, lipids and organelles. Several recent in instances in which fragmentation is accompanied by studies have shown that oocyte survival is controlled by a cleavage arrest (Jurisicova et al. 1996) or the complete number of cell death regulatory molecules responsible for destruction of all blastomeres (Yang et al. 1998). It is clear either activation or suppression of cell death (reviewed by that there are several time points when in vitro produced Tilly (2001)). It is also evident that deposition of maternal embryos are particularly susceptible to cellular fragmenta- products in the form of transcripts, proteins and organelles tion, arrest and apoptosis. This is true not only for humans, dramatically influences the success of preimplantation but also for bovines, as recent data confirmed that the embryo development (Muggleton-Harris & Brown 1988, onset of apoptosis seems to be developmentally regulated Gandolfi & Gandolfi 2001). As many cell survival/death in a stage-specific manner, but discrete features of the regulatory molecules are among these maternally stored apoptotic process may be differentially regulated and transcripts in both mice and humans (Jurisicova et al. independently modulated by the mode of embryo pro- 1998b, Exley et al. 1999, Metcalfe et al. 2004), it is likely duction (Gjorret et al. 2003). The underlying molecular that they may contribute to developmental competence of mechanisms responsible for these embryonic defects a zygote. Cell survival molecules, when inadequately remain to be determined. accumulated during oogensis (either too few suppressors A further phenomenon observed in embryos in almost or too many activators; Fig. 3), or change during the age- all mammalian species is that of developmental arrest. ing of oocytes in vitro (Metcalfe et al. 2003) or in vivo (Jur- The mechanisms leading to developmental arrest are isicova et al. 2002), may lead to survival disequilibria, unclear, yet, interestingly, the embryonic stage at which resulting in zygote or early embryo demise. transcription-dependent proteins are first detected in the The ability to pass through the transition to zygotic con- embryo coincides with the peak incidence of cleavage trol in vitro is believed to be a function of the cytoplasmic arrest in most species, suggesting that cleavage arrest components of the oocyte and is not a reflection of the could also be caused by a failure or delay in the onset of newly formed embryonic nucleus (Goddard & Pratt 1983). transcription (Telford et al. 1990). One cause of develop- There must therefore be a cytoplasmic component(s) that mental arrest may be chromosomal abnormalities (Bongso supports development through the blocking stage and this et al. 1991, Munne et al. 1995), which could contribute component(s) must be lacking or non-functional in those to a disruption in mRNA transcripts and ensuing protein embryos that do arrest. At the present time it is unclear synthesis, leading to interference with normal embryo which components of the ooplasm are responsible for this development (Devreker & Englert 2000). There is no rescue effect, but in recent studies ooplasmic injection of simple explanation for developmental arrest, as there is a isolated mitochondria was sufficient to rescue oocyte frag- broad spectrum of cellular defects that occur during mentation in mice (Perez et al. 1999, Acton et al. 2001), cleavage arrest, such as redistribution of mitochondria making these cellular organelles likely candidates for (Muggleton-Harris & Brown 1988, Acton et al. 2004) and mediating the positive action of transplanted ooplasm. abnormal subcellular localization of some proteins (Oha- shi et al. 2001), including Bcl-2 family members (Jurisi- cova et al. 2003). Genetic regulation of cell death in embryonic development Programmed cell death is a precisely coordinated set of Maternal effects events dependent upon the actions and interactions of at Progression of fertilized mammalian oocytes through clea- least 100 gene products that either repress or activate the vage, blastocyst formation and implantation is dependent process of cellular self-destruction (for reviews, see Green on the successful implementation of specific genetic and & Reed (1998), Vaux & Korsmeyer (1999), Adams & Cory developmental programmes. It is not surprising that varia- (2001)). The most widely studied of these genes are mem- bility in oocyte quality has a profound impact on develop- bers of the Bcl-2 family, which can be subdivided into mental competence and contributes to the high incidence two groups: cell death suppressors (Bcl-2, Bcl-xL, Bcl-w, of embryonic wastage observed during IVF procedures, as Mcl-1, A1) and cell death inducers (Bax, Bak, Mtd, BH3- it was estimated that the factors predisposing an embryo only proteins) (Fig. 2). Cell death inducers can be further to arrest are determined at or even before the zygote stage split into multidomain (containing BH1, BH2 and BH3 (Hardy et al. 2001). These factors are also regulated by domains such as Bax, Bak, Mtd) and single domain (con- maternal age, as increased frequencies of embryo frag- taining only BH3 domains, such as Bad, Bim, Hrk, Bid). mentation were observed during early cleavage stages in Members of this gene family act through a complex www.reproduction-online.org Reproduction (2004) 128 281–291

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Figure 2 Interactions between Bcl-2 family members. Three main subfamilies of the Bcl-2 family are represented: pro-apoptotic BH3 proteins (range of orange squares), anti-apoptotic proteins (range of green circles) and pro-apoptotic channel forming proteins (range of blue ovals). The changes in shading and colour gradient indicate differences in binding partners and affinities between members in the pro-apoptotic subfamilies with members of the antiapoptotic subfamily. Proapoptotic BH3 proteins: Puma and Bim bind to all antiapoptotic members equally; Bik, Hrk and Bid bind to Bcl-xL, Bcl-w and Bf1/A1; Bmf and Bad bind to Bcl-2, Bcl-xL and Bcl-w; Noxa binds to Bf1/A1 and Mcl-1. Proapoptotic channel forming proteins: Bax and Bak bind to Bcl-2, Bcl-xL and Bcl-w, whereas Mtd/Bok binds to Bf1/A1 and Mcl-1. network of homo- and heterodimers (Adams & Cory represents a single oocyte (Fig. 3). According to the pat- 1998). The fate of the cell is determined by the ability of tern of expression profiles, the oocytes were grouped into the cell death suppressors to sequester, and thus neutral- three categories: those shifted towards cell death as a ize, the actions of the cell death inducers. Hence, the result of an imbalance shifted towards pro-apoptotic relative concentration of pro- and anti-apoptotic proteins determines whether a cell lives or dies (Antonsson & Martinou 2000). Many Bcl-2 family members localize permanently or transiently to mitochondrial membranes, where they are believed either to regulate, or to form, mitochondrial pores. Opening of these pores results in changes in mitochondrial membrane potential, accompanied by the release of several death-inducing factors from the mitochondrion into the cytoplasm. On the basis of research on apoptosis in different cell types using various cell death triggers, it has become clear that there are at least two independent pathways leading to activation of apoptosis: intrinsic, mitochond- rially driven, and extrinsic, without mitochondrial involve- ment. These pathways are not completely independent, as cross-talk and an augmentation loop exist between the intrinsic and extrinsic pathways (for reviews see Desagher & Martinou (2000), Hengartner (2000)). The finite control Figure 3 Distribution of five pro- and anti-apoptotic genes in 15 indi- of cell death seems to be highly regulated by the interplay vidual MII stage human unfertilized oocytes obtained from several between Bcl-2 family members, and these interactions are patients. The expression of Bag-1 (yellow), Bax (orange), Bcl-x (pale blue), Hrk (red) and Mcl-1 (dark blue) was determined using quantitat- purported to have an important role in embryo survival. ive dot–blot RT-PCR. The total amount of gene expression was calcu- The relative rates of expression of the genes of several lated for these pro-apoptotic (orange and red) and anti-apoptotic Bcl-2 family members in human MII oocytes were (yellow, pale blue and dark blue) genes, each pie piece representing assessed through quantitative dot–blot RT-PCR, including the contribution for that particular gene. The oocytes were then pro-apoptotic members Bax and hara-kiri and anti-apop- grouped on the basis of their expression profiles. (A) Oocytes with a totic members Bag-1, Bcl-x and Mcl-1. From the total high level of pro-apoptotic gene expression, possessing an imbalance towards programmed cell death. (B) Oocytes with almost equal distri- amount of gene expression in each oocyte, the contri- bution of pro- and anti-apoptotic factors suggestive of a balance bet- bution from each family member was established and ween survival and death molecule contributions. (C) Oocytes with an the results presented as pie graphs, in which each graph excess of anti-apoptotic factors indicative of a strong survival pathway.

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Downloaded from Bioscientifica.com at 10/01/2021 09:50:56PM via free access PCD gene expression in human preimplantation embryos 285 genes, those predisposed to survival because of a strong Channel-forming pro-apoptotic family members level of anti-apoptotic gene expression, and those with a Expression of the pro-apoptotic molecule Bax has been balance in expression of pro- and anti-apoptotic genes. studied frequently and this gene appears to be consistently These results suggest that there are inherent differences in expressed throughout preimplantation development, with the level of gene expression for these gene families, and only a marginal increase in accumulation of the protein in that the balance between the expression of pro- and anti- poor quality embryos (Spanos et al. 2002, Metcalfe et al. apoptotic genes may be a central determinant of embryo 2004). Interestingly, at least one study described Bax survival. immunoreactivity to be restricted to polarized domains of some cellular fragments (Antczak & Van Blerkom 1999), Anti-apoptotic Bcl-2 family members suggesting that this protein may participate in the restric- tion of subcellular compartments and possibly the removal This group of proteins are the most widely studied genes of unwanted or harmful organelles or other cytoplasmic during both oocyte and preimplantation embryo develop- components. It is now known that both Bax and Bad are ment and consists of several functionally overlapping postranslationally modified. Bad, having high binding affi- homologues. Bcl-2, a founding member of this pro-survi- nity for Bcl-xL and Bcl-2, displaces Bax from Bcl-x/Bax val gene family, had been reported to be expressed by complexes and allows Bax to homodimerize, change con- many (Spanos et al. 2002), but not all, human embryos formation, translocate to mitochondria and induce cell before the four-cell stage (Liu et al. 2000, Metcalfe et al. death (Capano & Crompton 2002). Bad itself is regulated 2004). Immunolocalization confirmed expression of Bcl-2 by phosphorylation, which prevents its binding to Bcl-xL, in both good and poor quality embryos, but a subtle and increases the affinity of Bad for the 14-3-3 sequester- increase in accumulation of this protein was observed in ing protein, the binding of which inactivates the pro-apop- the cellular fragments of some embryos (Spanos et al. totic potential of Bad (Zha et al. 1996). It remains to be 2002, Metcalfe et al. 2004). determined whether Bad and Bax are expressed during In our hands, Mcl-1 is the most abundantly and consist- preimplantation development in their active or inactive ently expressed Bcl-2 family member detected during the conformation, and if these post-translational modifi- early cleavage stages (Jurisicova et al. 2003). These data cations, rather than transcriptional regulation, may explain are partially consistent with those of Metcalfe et al. the regulation of cellular fragmentation. (2004), who reported more variable expression between Two other channel-forming pro-apoptotic molecules, two- to four-cell stages, followed by equally variable Mcl- Bak and Bok/Mtd, have only recently been assessed 1 protein localization at the blastocyst stage. The lack of (Metcalfe et al. 2004). Both are expressed, albeit more Mcl-1 expression observed in human blastocysts is intri- variably than Bax, throughout preimplantation develop- guing, as Mcl-1 is the only pro-survival Bcl-2 family mem- ment, Bok being more abundant during early and Bak ber of which the disruption results in preimplantation during later embryonic stages. Interestingly, preliminary embryonic lethality. data suggest that fragmentation at the four-cell stage may Another important family member likely to regulate suc- be associated with increased expression of Bak (Metcalfe cessful progression through preimplantation embryo et al. 2004); however, this needs to be confirmed by development is Bcl-x. Although its RNA is less abundant immunolocalization. than that of Mcl-1 (Jurisicova et al. 2003, Metcalfe et al. 2004), its protein is expressed in high levels during both BH3 family members early and late cleavage stages (Antczak & Van Blerkom 1999, Metcalfe et al. 2004). Using an antisense mRNA Very little is known about the expression of BH3-only approach in murine embryos, we observed that the classi- molecules during preimplantation embryo development. cal Bcl-xL isoform is not required for progression of eight- This is clearly an unexplored field for new research, as cell embryos through the blastocyst stage. In contrast, up- these are the molecules that are the initiators of cell survi- regulation of the alternative splicing form Bcl-xS renders val decisions and are responsive to cellular needs and blastomeres more susceptible to death and cell cycle requirements. Also, many of these genes are transcription- arrest (Jurisicova et al. 2001), as previously observed in ally regulated (Puthalakath & Strasser 2002), making them other cell types (Fridman et al. 1998, Lindenboim et al. the most suitable and meaningful candidates for detection 2000), suggesting that this gene may be an important via RT-PCR expression studies, a frequently used method component of the regulation of embryonic fate. Among for studying preimplantation embroyo expression profiles. other anti-apoptotic Bcl-2 family members, Bcl-w and Bfl- From the few genes that have been studied, it is appar- 1/A1 are only weakly and inconsistently expressed (Jurisi- ent that BH3 family members have a developmentally cova et al. 1998a, Metcalfe et al. 2004) and thus are unli- regulated pattern of expression, with Bid being the least kely to play a part in regulating early embryo demise. frequently expressed (Metcalfe et al. 2004). Both Bik and Moreover, their disruption in mouse does not interfere Bad are abundant during both early and later preimplanta- with progression of embryonic development (Print et al. tion stages, with some subtle differences between frag- 1998, Ross et al. 1998). mented and non-fragmented embryos (Metcalfe et al. www.reproduction-online.org Reproduction (2004) 128 281–291

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2004). These results contrast with the lack of Bad fragments was increased in embryos with multinucleated expression by early cleavage stage embryos previously blastomeres and this was unrelated to embryo morphology reported by Spanos et al. 2002) who detected Bad (Martinez et al. 2002). expression by RT-PCR in very few two- to four-cell These data also agree with reports by Spanos et al. embryos (two out of nine), but frequently around the time (2002) and Van Blerkom et al. (2001), as in those studies of compaction and at the blastocyst stage. caspase activity was observed only in arrested embryos or We have recently obtained descriptive correlative data after compaction at the morula and blastocyst stages, fre- suggesting that Hrk may be involved in the regulation of quently associated with apoptotic nuclei (Spanos et al. survival of preimplantation human embryos, as both its 2002). The group of arrested and fragmented embryos stu- transcript and its protein concentrations were increased in died by our laboratory showed that active caspase 3/7 like a subset of arrested fragmented four- and eight-cell activity was associated with cellular fragments containing embryos (Jurisicova et al. 2003). It remains to be estab- nuclear material or in blastomeres with condensed chro- lished what regulates the expression of this gene in preim- matin, while the caspase 3 transcript was detected at all plantation embryos and how this relates to embryo demise. embryonic stages, and even increased in some fragmented embryos (Jurisicova et al. 2003). Other studies, however, Extrinsic pathway have failed to demonstrate a strong correlation between caspase activation and embryo fragmentation. Attempts to Involvement of the extrinsic pathway in the regulation of reduce murine embryo fragmentation with caspase inhibi- human preimplantation embryo development has been tors (Xu et al. 2001) and a failure consistently to detect investigated by two groups. Kawamura et al. (2001) apoptotic markers in cytoplasmic embryo fragments from reported that human embryos at the two- and four-cell human embryos (Antczak & Van Blerkom 1999, Spanos stage express Fas ligand, but this was not observed by Liu et al. 2002) suggests that caspase independent mechanisms et al. (2000), as Fas ligand transcript was not detected in may play a part in this process, as has been observed in any of the viable or fragmented embryos they studied. some cell types (Donovan & Cotter 2004). Interestingly, the report by Kawamura et al. (2001) suggested a bimodal switch in death receptor expression between the two- and four-cell stages. Tumour necrosis Mouse models of early embryo loss factor receptor was detected at the two-cell stage, but Fas was detected only at the four-cell stage, and cellular frag- Animal models have a key role in investigations of the pro- mentation did not alter its expression profile. These two cess of embryogenesis, in which murine embryos have reports describe contradictory results and further investi- been extensively studied in an effort to elucidate pathways gation into the role of extrinsic signals in preimplantation and mechanisms common across mammalian species. embryo cell death is required. Comparative gene expression studies of mouse and human preimplantation embryo development have identified strik- Caspase requirement for cellular arrest and ing similarities in the conservation of the molecular and fragmentation cellular framework and in the execution of developmental decisions, making the mouse an excellent model for the Caspases are a family of cysteine-dependent aspartate- study of human development. The mouse model is further directed proteases that are present in an inactive zymogen attractive as it allows for easy genetic manipulation and form before being cleaved to an active form. Upon acti- induction of various pathological conditions. vation, caspases cleave a variety of intracellular polypep- tides, including, but not limited to, structural elements of the cytoplasm and nucleus. This leads to disruption of sur- Zygote arrest gene vival pathways and the disassembly of important architec- Zygotic arrest gene (ZAR) is a putative transcription activa- tural components of the cell, contributing to the well tor with a prototypical PHD domain. Female mice carry- documented morphological and biochemical changes that ing two disrupted ZAR copies are infertile as a result of characterize apoptotic cell death (Earnshaw et al. 1999). the inability of the embryo to proceed through the first Caspases had been extensively studied, and yet their role cleavage division. Cleavage arrest is followed by cyto- in early embryo development remains unclear. Current plasmic fragmentation and complete disintegration of the studies have used fluorescent caspase substrates, which embryo (Wu et al. 2003). The functional importance of become trapped upon activation within a cell. Although a ZAR in the human is currently unclear, but may be clini- broad range of caspase inhibitors are commercially avail- cally inconsequential, as zygote arrest is rare in the able, no systematic studies beyond the use of caspase 3/7 human and accounts for less than 1% of embryonic loss. substrates have been reported. Martinez et al. (2002), who used the broad range caspase substrate, FITC-VAD, Mater detected active caspases frequently in cellular fragments, but activity was only occasionally found in blastomeres of Mater is a protein that was originally identified in a screen poor quality embryos. The percentage of caspase-positive for genes responsible for autoimmune oophoritis (Tong

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Downloaded from Bioscientifica.com at 10/01/2021 09:50:56PM via free access PCD gene expression in human preimplantation embryos 287 et al. 2000b). On the basis of a sequence homology, this may not act through the classical apoptotic pathway. The oocyte-specific product belongs to a newly discovered second, more subtle, phenotype relates to embryonic group of NALP proteins (Tschopp et al. 2003). The arrest, as embryos flushed at earlier stages arrest shortly majority of NALP proteins are adaptors involved in the after being placed in culture (Rinkenberger et al. 2000). regulation of inflammation and have a marginal effect on This response is probably a result of embryonic stress the regulation of apoptosis, but the precise function of encountered during in vitro culture. These findings clearly Mater remains unclear. Oocytes ovulated by females car- indicate an involvement of Mcl-1 in the regulation of cell rying a Mater mutation are capable of fertilization and cycle progression and place this molecule among a hand- proceed through the first cleavage; however, this is fol- ful of proteins functionally important in the developmental lowed by arrest and cellular fragmentation. Activation of decisions made by the early embryo. the embryonic genome is impaired, accompanied by a decrease in protein synthesis (Tong et al. 2000a). A recent Defender against apoptosis-1 report on the localization of Mater protein revealed that its primary site is in the mitochondrion and nuclear envel- Defender against apoptosis-1 (Dad-1) is an evolutionary opes (Tong et al. 2004). The specific function of Mater highly conserved enzyme, which catalyses the transfer protein and its relevance to the quality of human embryos of preassembled high mannose oligosaccharides onto remains to be established. specific asparagine residues of nascent polypeptides (Sanjay et al. 1998). This enzyme is capable of interacting with and modifying Mcl-1 (Makishima et al. 2000). Thus it Heat shock factor-1 is not surprising that disruption of this gene leads to Heat shock factor (HSF)-1 is a protein responsible for the embryonic lethality at the blastocyst stage (Nishii et al. induction of expression of the heat shock protein (HSP) 1999, Brewster et al. 2000), phenocopying the Mcl-1 gene under both physiological and cell stress conditions. knockout mouse. Dad-1 mutants, however, display an HSPs have numerous roles in cell function, including increased rate of cell death in the inner cell mass both in modulation of protein activity, regulation of protein degra- vitro and in vivo, suggesting that this enzyme may have dation and transport across organelles, in addition to modification targets other than Mcl-1. ensuring correct protein folding (Takayama et al. 2003). More recently, HSPs were implicated in the regulation of Bax and p53 cell death, as Hsp 70 had been shown to prevent both Disruption of Bax and p53 does not result in any known caspase dependent and caspase independent cell death developmental abnormalities observed during the preim- (Ravagnan et al. 2001). In addition, Hsp 27 is capable of plantation period under physiological conditions, but their interfering with cytochrome-c mediated apoptosome acti- embryonic phenotypes appear more obvious upon chal- vation, leading to inhibition of the mitochondrial death lenge. Lack of either of these genes provides a rescue of pathway (Bruey et al. 2000). Both of these HSPs are regu- blastocysts from cell death induced by hyperglycaemia lated by HSF-1 (Sistonen et al. 1994, Cooper et al. 2000). (Moley et al. 1998, Keim et al. 2001). Suppression of HSF-1 is expressed in high levels during preimplanta- hyperglycaemia-induced cell death is dependent on both tion embryo development (Christians et al. 1997). Interest- Bax and p53, and is accompanied by a decrease in the ingly, embryos lacking a functional maternal copy of this number of embryo resorptions, directly implicating cell gene during oocyte development arrest most frequently death in postimplantation embryonic loss (Moley 2001). between the one- and three-cell stages, followed by par- tial cellular fragmentation. Zygote development is unim- Survivin paired and is followed by a delay in the onset of embryonic genome transcription (Christians et al. 2000). Survivin, an IAP family member (Vaux & Silke 2003a, b), The connection of HSF-1 with other established cell death has a pattern of expression restricted to a subset of tissues pathways in these embryos has not been explored. including gametes, embryos and some cancers (Kobayashi et al. 1999). Depending on the cell type and conditions used, this gene has been implicated both in the regu- Myeloid cell leukaemia-1 lation of cell cycle progression and in the inhibition of Myeloid cell leukaemia-1 (Mcl-1) is the only pro-survival apoptosis (Altieri 2003). Disruption of survivin leads to gene the disruption of which in the mouse leads to early embryonic lethality between the morula and blastocyst embryonic lethality. Blastocysts lacking Mcl-1 have been stages, leading to formation of giant nuclei, decreased detected at day 4 in the reproductive tract, but these cell number, impaired mitotic figures (Uren et al. 2000) embryos failed to implant and were also unable to pro- and increased apoptosis (Kawamura et al. 2003). Thus duce viable blastocyst outgrowths in vitro (Rinkenberger the mode of death in these embryos is more consistent et al. 2000). No increase in apoptosis was observed in with mitotic catastrophe, which can not be rescued by these blastocysts and the embryo loss could not be res- inactivation of caspases or other cell death genes (Okada cued by disruption of Bax or p53, suggesting that Mcl-1 et al. 2004). At the present time it is unknown whether www.reproduction-online.org Reproduction (2004) 128 281–291

Downloaded from Bioscientifica.com at 10/01/2021 09:50:56PM via free access 288 A Jurisicova and B M Acton alterations in the expression of survivin can contribute particular interest to apoptosis is the polarized distribution to the broad range of nuclear defects described in of Bax, Bcl-x and Hrk, indicating that temporal changes in human embryos. gene expression alone cannot be used to predict apopto- sis, as spatial expression may also play a significant part. It Endonuclease G is therefore feasible to propose that spatial localization Endonuclease G is a recently identified nuclear-encoded could be an adaptive mechanism of the oocyte or embryo mitochondrial protein, reported to be important for both to sequester certain gene products. The formation of nuclear DNA fragmentation during apoptosis and mito- embryo fragments could be a survival mechanism in chondrial DNA replication (Li et al. 2001). On induction which pro-apoptotic gene products are removed from the of apoptosis, it is released from the mitochondria and embryo in an attempt to rescue the embryo from the translocates to the nucleus, where it is involved in the oli- damaging effects of an imbalance in pro- and anti-apopto- gonucleosomal degradation of DNA through both caspase tic gene products. Alternatively, as a result of fragmenta- dependent and caspase independent mechanisms (Lor- tion and selective removal of cytoplasm, an imbalance in enzo & Susin 2004). Disruption of the EndoG gene results protein expression in the affected blastomeres could result in embryonic lethality during the morula to blastocyst in the induction of programmed cell death pathways transition (Zhang et al. 2003). Heterozygote animals show (Antczak & Van Blerkom 1999). Moreover, this process distinct phenotypes that could be attributed to a cell death could be mediated by caspase, as caspase-positive frag- imbalance, but oocytes from these females contained a ments have been observed to detach from healthy blasto- similar number of mitochondria compared with wild-type meres that subsequently underwent mitotic division in controls, suggesting that a single copy of EndoG is suffi- culture (Martinez et al. 2002). If attempted rescue is suc- cient to maintain mitochondrial DNA replication. These cessful and the remaining blastomeres are not further chal- findings indicate that EndoG is essential during early lenged, embryo cleavage and development will proceed. embryogenesis, but its relationship to human embryo If this rescue attempt fails and the remaining blastomeres wastage has not yet been investigated. experience other setbacks, the embryo will undergo arrest, followed by the execution of a caspase dependent apopto- tic programme in the remaining blastomeres. Conclusions and perspectives Secondarily, we would like to propose that embryonic From all the references cited, it is clear that genes govern- arrest in vitro is accompanied by cellular events that are ing cell death pathways are involved in the regulation of partially consistent with programmed cell death type II or preimplantation embryo survival. This is evident, not only an autophagic mode of death (Levine & Klionsky 2004). It from descriptive observational studies, but also from func- appears that, under certain conditions, when the classical tional interventional animal experiments. Although some apoptotic programme is heavily suppressed, cells can pro- types of cell death in preimplantation embryos, especially long their survival by using this catabolic pathway to pro- during the postcompaction stages, are characteristic of the duce energy, and only upon complete exhaustion of their classical apoptotic mode of death, others display mor- intracellular energy supply do they succumb to autopha- phologies that are not fully consistent with classical apop- gic death (Gozuacik & Kimchi 2004). The morphological tosis. To complicate the issue further, added to this is the hallmark of arrested embryos observed in vitro – the par- difficulty in obtaining high quality, developmentally com- tial shrinking of blastomeres and formation of cytoplasmic petent human embryos for study. It is clear that the vesicles – may be the result of an attempt of the cells to majority of human embryos donated for research are suf- save themselves by catabolizing their own cytoplasm. fering from various cellular defects, including abnormal Thus, if the embryo possesses a battery of transcripts that nuclear ploidy, altered genomic imprinting and variable would strongly promote survival but finds itself in responses to culture conditions, and thus are likely to be a inadequate culture conditions, it may trigger the autopha- poor reflection of the true molecular pathways found in gic mode of death. The partial destruction of mitochon- healthy embryos. Further studies with appropriate controls dria, a process regulated by the Bcl family members via comparing ‘good’ with ‘poor’ quality embryos are needed an autophagic mode of cell death (Tolkovsky et al. 2002), to provide an accurate indication of preimplantation could also contribute to embryo demise. In contrast, an embryo development. embryo originating from an oocyte with imbalanced cell Current studies support the hypothesis that cellular frag- death machinery or a zygote that fails to execute basic mentation in a subset of human preimplantation embryos embryonic decisions would opt for the rapid apoptotic could be regulated by certain components of a genetic mode of death, accompanied by cellular fragmentation. programme of cell death. As blastomeres are among the These two modes of cell death may be intertwined and biggest cells in the human body, we speculate that they only further explorations of the genetic pathways involved utilize some of the cell death machinery to remove the in the regulation of death decisions will establish if these areas of cytoplasm, also called ‘highly polarized domains’, modes of death are truly genetically regulated and that may prove detrimental for future development, as relevant during mammalian preimplantation embryo proposed previously (Antczak & Van Blerkom 1999). Of development.

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