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 Reproduction (2004) 128 281–291 www.reproduction-online.org 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