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BOR - Papers in Press, published online ahead of print May 5, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026963
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BIOLOGY OF REPRODUCTION 71, 871–877 (2004)
DOI: 10.1095/biolreprod.103.026963
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

TRAIL and KILLER Are Expressed and Induce Apoptosis in the Murine Preimplantation Embryo1

Joan K. Riley3, Jennifer M. Heeley3, Amanda H. Wyman3, Elizabeth L. Schlichting3, and Kelle H. Moley2,3,4

Department of Obstetrics and Gynecology3 Department of Cell Biology and Physiology,4 Washington University School of Medicine, St. Louis, Missouri 63110


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand) and KILLER are a death-inducing ligand and receptor pair that belong to the TNF and TNF-receptor superfamilies, respectively. To date, only one apoptosis-inducing TRAIL receptor (murine KILLER [MK]) has been identified in mice, and it is a homologue of human Death Receptor 5. Whereas the expression of other death receptors, such as Fas and TNF receptor 1 have been documented in mammalian preimplantation embryos, no evidence currently demonstrates either the presence or the function of TRAIL and its corresponding death receptor, MK. Using reverse transcription-polymerase chain reaction and confocal immunofluorescent microscopy, we found that both TRAIL and MK are expressed from the 1-cell through the blastocyst stage of murine preimplantation embryo development. These proteins are localized mainly at the cell surface from the 1-cell through the morula stage. At the blastocyst stage, both TRAIL and MK exhibit an apical staining pattern in the trophectoderm cells. Finally, using the TUNEL assay, we demonstrated that MK induces apoptosis in blastocysts sensitized to TRAIL via actinomycin D. Taken together, these data are the first to demonstrate the presence and function of TRAIL and MK, a death-inducing ligand and its receptor, in mammalian preimplantation embryos.

apoptosis, cytokines, early development, embryo, immunology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis, or programmed cell death, plays a crucial role in normal embryo development by eliminating both unnecessary and abnormal cells [1]. Cellular stresses, such as hyperglycemia, have been shown to induce apoptosis in preimplantation embryos [2, 3]. It is hypothesized that apoptosis induced by hyperglycemia during the preimplantation period may, in part, lead to the increased incidence of malformations and congenital defects seen among the infants of women with diabetes [4–6]. Determining the mechanisms by which preimplantation embryos undergo apoptosis therefore may be important to understanding the deleterious effects of certain cellular stresses on embryo development.

Apoptosis can be triggered by one of two cell death pathways termed the intrinsic and extrinsic pathways. The intrinsic, or mitochondrial, pathway can be activated by numerous cellular stresses, whereas the extrinsic cell death pathway is triggered by death receptors belonging to the tumor necrosis factor (TNF)-receptor superfamily [7]. TRAIL (TNF-related apoptosis-inducing ligand) is an apoptosis-inducing member of the TNF ligand superfamily that is expressed in most human and mouse tissues [8, 9]. TRAIL induces apoptosis by binding to its death receptor, which is also widely expressed [7, 10]. Humans have five TRAIL receptors, only two of which induce apoptosis [11]. To date, only one apoptosis-inducing TRAIL receptor has been identified in mice, and it is a homologue of human Death Receptor 5 (DR5)/KILLER [10]. In addition, three novel murine TRAIL receptors were recently identified. These receptors, mDcTRAILR1 and mDcTRAILR2 (the latter consists of two splice variants), are proposed murine decoy receptors, because they bind TRAIL but do not transduce an apoptotic signal [12]. Moreover, it was demonstrated that mTRAIL also binds to the soluble TNF receptor-homologue osteoprotegerin. Thus, to date, five murine TRAIL receptors are known.

The binding of TRAIL to murine KILLER (MK) induces apoptosis in both human and mouse cell lines [10]. In humans, apoptosis-inducing TRAIL receptors contain a cytoplasmic death domain, which recruits the adapter-molecule FADD to the receptor. In turn, FADD recruits procaspase-8 to this complex, resulting in caspase-8 activation and the subsequent activation of downstream effector caspases, thus leading to programmed cell death [13–15].

Although TRAIL and MK expression have not been characterized in mammalian preimplantation embryos, the expression and function of this receptor-ligand pair has been explored in the reproductive system of animals at times other than preimplantation embryo development. It was shown that the avian TVB (DR5-like) receptor is expressed and induces apoptosis in chick embryo fibroblasts [16]. Moreover, TRAIL and MK may be involved very early in the reproductive process, during oocyte development, because TVB is expressed in hen granulosa cells [17]. This observation also extends into mammalian systems, because both DR4 and DR5 homologues and TRAIL are expressed in porcine ovaries [18, 19]. It is hypothesized that TRAIL and its porcine death receptors are involved in granulosa cell apoptosis during atresia and that DcR1 inhibits this process [20]. TRAIL and its receptors may also play a role later in the reproductive process, because they are present in human placenta and, therefore, may be important to reproductive success [21, 22]. Thus, TRAIL and its corresponding death receptors may be involved in the induction of apoptosis at various stages during the reproductive process.

The aforementioned work characterizing the expression and potential function of TRAIL and its death receptors during reproduction led us to examine whether this death-inducing ligand and receptor are expressed and induce apoptosis in murine preimplantation embryos. Identification of the presence and functionality of TRAIL and MK on preimplantation embryos may prove to be important for elucidating the mechanism by which some cellular stresses induce apoptosis in early mammalian embryos.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Embryo Recovery and Culture

Embryos were recovered as previously described [23]. In short, 3-wk-old female mice (B6 x SJL F1; Jackson Laboratories, Bar Harbor, ME) were given free access to food and water and were maintained on a 12L: 12D photoperiod. Female mice were superovulated with an i.p. injection of 10 IU of eCG (Sigma, St. Louis, MO), followed 48 h later by 10 IU of hCG (Sigma). Female mice were mated with males of proven fertility overnight following the hCG injection. Mating was confirmed by identification of a vaginal plug.

Mice were killed at 24, 48, 54, 72, or 96 h post-hCG injection to recover embryos at the 1-cell, 2-cell, 4-cell, morula, and blastocyst stages, respectively. Embryos were recovered by flushing dissected uterine horns and ostia with human tubal fluid (HTF) medium (Irvine Scientific, Santa Ana, CA) containing 0.25% BSA (fraction V; Sigma). One-cell embryos were immediately placed in HTF medium containing 0.3 mg/ml of hyaluronidase (Sigma) to remove the cumulus cells. Embryos were then rinsed in HTF without BSA and either used immediately for RNA isolation or fixed for staining. Blastocysts used in the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were harvested 96 h post-hCG injection and subsequently incubated in vitro for an additional 4 h in HTF alone or in HTF supplemented with 5 µg/ml of recombinant murine TRAIL (Biomol, Plymouth Meeting, PA), 0.5 µg/ml of actinomycin D (Sigma), or a combination of TRAIL plus actinomycin D. All procedures described above were reviewed and approved by the animal studies committee at Washington University and were performed in accordance with Institutional Animal Care and Use Committee approval.

Reverse Transcription-Polymerase Chain Reaction and Nested Polymerase Chain Reaction

The RNA was isolated from embryos using the RNeasy Mini Kit (Qiagen, Valencia, CA). The RNA at each stage of development was isolated from a single pool of embryos, which consisted of 300 one-cell embryos, 150 two-cell embryos, 75 four-cell embryos, 20 morulas, and 5 blastocysts. The RNA isolated from each pool was then divided into four separate polymerase chain reaction (PCR) reactions. TRAIL primers were added to two of the reactions, and MK primers were added to the other two reactions. One tube within each pair was used as a negative control and did not contain reverse transcriptase. The reverse transcription (RT)-PCR was not quantitative. The sequences of the PCR primers are listed in Table 1.


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  		TABLE 1. RT-PCR and nested PCR conditions, including primer sequences, expected product sizes, annealing temperatures, and number of cycles

The RT-PCR reaction was carried out using the Titan One Tube RT-PCR System (Roche, Basel, Switzerland). In brief, the extracted RNA was added to a mixture containing 200 µM dNTPs (Invitrogen, Carlsbad, CA), 0.4 µM sense and antisense primers (Integrated DNA Technologies, Coralville, IA), 5 mM dithiothreitol, 5 U of RNase Inhibitor (Roche), 1 µl of dimethyl sulfoxide (DMSO; Sigma), 1x RT-PCR buffer, and 0.5 µl Enzyme Mix in a 50-µl total reaction volume. The RT-PCR negative-control reactions contained 0.5 µl of DNA polymerase from the Expand High-Fidelity PCR System (Roche) in place of the Enzyme Mix, which contains AMV reverse transcriptase. The RT-PCR program was as follows: 50°C for 30 min, 94°C for 2 min, 94°C for 30 sec, 52°C for 30 sec, and 68°C for 1 min. The PCR program contained 50 cycles, followed by a 7-min extension at 68°C, and was performed in a PTC-100 Peltier Thermal Cycler (MJ Research, Inc., Waltham, MA).

A nested PCR was performed on each RT-PCR reaction. Two microliters of the original RT product were added to a mixture containing 200 µM dNTPs, 0.4 µM sense and antisense primers, 1 µl of DMSO, 1x ThermoPol Reaction Buffer, and 1 µl of Taq DNA Polymerase (New England Biolabs, Beverly, MA) in a 100-µl total reaction volume. The PCR program was as follows: 94°C for 2 min, 94°C for 30 sec, 52°C for 30 sec, and 72°C for 1 min. The PCR program contained 50 cycles, followed by a 7-min extension at 72°C.

The PCR products were run on a 1% agarose gel (Invitrogen) containing ethidium bromide (Fisher, St. Louis, MO) and visualized with an AlphaImager 2200 (Alpha Innotech Corp., San Leandro, CA). One microliter of each nested PCR product was cloned into the pCR2.1 vector using the TA Cloning Kit (Invitrogen). The inserts were subsequently sequenced according to the Big Dye protocol (Applied Biosystems, Foster City, CA) with T7 and M13 reverse primers. The PCR products were analyzed with an ABI Sequencer 3730 (Applied Biosystems).

Immunofluorescent Staining

Immunofluorescent staining techniques have been described for embryo preparations previously [23]. All labeling was performed in microdroplets. Embryos were fixed in 3% paraformaldehyde for 20 min and then permeabilized in either 0.1% Tween-20 or 0.1% Triton for 30 min. The embryos were subsequently blocked for 1 h with 20% normal goat serum (Pierce, Rockford, IL) in PBS containing 2% BSA (PBS/BSA). Embryos were then washed three times for 10 min each in PBS/BSA and incubated for 40 min in 20 µg/ml of one of the following antibodies: anti-TRAIL (H-257; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-DR5 (Sigma), or anti-Glut-4 (gift of Dr. Mike Mueckler, Washington University, St. Louis, MO). The embryos were then washed three times for 10 min each in PBS/BSA and incubated with the secondary antibody, Alexa Fluor 488 goat anti-rabbit immunoglobulin G (Molecular Probes, Eugene, OR) at a concentration of 2 µg/ml for 30 min followed by 4 µM To-Pro-3-iodide, a nuclear stain, for 20 min. Finally, the embryos were washed three times for 10 min each in PBS and mounted in drops of Vectashield (Vector Laboratories, Burlingame, CA) under a coverslip. Fluorescence was detected with a Nikon C1 laser-scanning confocal microscope (Nikon, Melville, NY). Confocal images were taken at 63x magnification. These experiments were preformed in triplicate with at least five embryos per group for each experiment.

TUNEL Assay

Apoptosis was examined using the TUNEL assay as previously described [2]. After the embryos were fixed and permeabilized as described above, apoptosis was assessed using the In Situ Cell Death Detection Kit (Roche) according to the manufacturer's protocol. The nuclei were stained and the embryos visualized as described above. A Z-series consisting of nine sections was taken for each embryo using a Nikon C1 laser-scanning confocal microscope. The total number of nuclei and apoptotic nuclei per embryo were quantitated manually for each Z-series. The sum of the apoptotic nuclei and the total nuclei within each treatment group was then utilized to calculate the percentage of TUNEL-positive nuclei per embryo.

Statistical Analysis

Differences between TUNEL values experienced by control, TRAIL-, actinomycin D-, and actinomycin D plus TRAIL-treated embryos were compared by an ad hoc ANOVA coupled with a Bonferroni/Dunn test using Statview 4.51 (Abacus Concepts, Inc., Piscataway, NJ). All data are expressed as the mean ± SEM. The TUNEL assay was performed in three independent experiments with at least seven blastocysts per group. A total of 22 blastocysts were examined for each treatment group over the course of the three experiments. Significance was defined as P < 0.01.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TRAIL and KILLER Are Expressed Throughout Preimplantation Embryo Development

Both TRAIL and MK RNA were detected in the murine 1-cell, 2-cell, 4-cell, morula, and blastocyst stages (Fig. 1). In contrast, no significant message was detected in negative-control reactions that lacked reverse transcriptase. All PCR products were cloned and sequenced to confirm their identity.



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  		FIG. 1. TRAIL and MK RNA expression in murine preimplantation embryos. The RNA was isolated from a single pool of embryos at the 1-cell, 2-cell, 4-cell, morula, and blastocyst stages of development. An RT-PCR followed by a nested PCR was performed on the RNA isolated at each stage to detect the expression of both TRAIL and MK message. A negative control that was run for each pool of RNA did not contain reverse transcriptase (–), whereas reverse transcriptase was added to the test reactions (+)

We next examined the protein expression patterns of the ligand and receptor (Fig. 2). Glut-4 is not expressed in murine preimplantation embryos [24, 25]; thus, an anti-Glut-4 antibody was used as a species-matched negative control. No Glut-4 expression was seen at any stage of development. Both TRAIL and MK protein were present from the 1-cell through the blastocyst stage of embryo development. These proteins were localized largely at the plasma membrane from the 1-cell through the morula stage. During this period, TRAIL and MK were also detected in the cytoplasm of the developing embryo. In contrast, at the blastocyst stage, TRAIL and MK exhibited a largely apical staining pattern, with cytoplasmic staining throughout the remainder of the embryo, including the inner cell mass. Thus, with respect to plasma membrane expression, TRAIL and MK only appear on the apical surface of trophectoderm cells. The localization patterns of these proteins during the preimplantation period indicate that TRAIL and MK expressed in the embryos are exposed to the maternal environment.



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  		FIG. 2. TRAIL and MK protein expression in preimplantation embryos. Embryos were stained with one of the following antibodies: anti-Glut-4, anti-TRAIL, or anti-DR5 (MK). The embryos were then incubated with a secondary antibody, Alexa Fluor 488 goat anti-rabbit immunoglobulin G (green fluorescence). To-Pro-3 iodide was used to stain the nuclei (blue fluorescence). This experiment was performed three times with at least five embryos per group for each experiment

MK Induces Apoptosis in Preimplantation Embryos when Triggered by Its Ligand TRAIL

To determine whether MK is a functional receptor able to induce apoptosis in preimplantation embryos, blastocysts were harvested and then cultured in vitro for an additional 4 h in media alone or in media supplemented with TRAIL, actinomycin D, or actinomycin D plus TRAIL. The embryos were subsequently analyzed via the TUNEL assay. Control embryos and embryos cultured in the presence of TRAIL or actinomycin D alone displayed very few apoptotic nuclei per embryo (3.2%, 2.4%, and 3.4%, respectively) (Fig. 3, red). In contrast, blastocysts cultured in a combination of TRAIL and actinomycin D displayed a significant increase in the percentage of apoptotic nuclei per embryo (8.0%) compared to controls. As previous reports have also shown, actinomycin D, a nucleic acid-synthesis inhibitor, can sensitize cells to TRAIL-induced apoptosis [26–30]. Therefore, MK is functional at the blastocyst stage and induces apoptosis on receptor ligation.



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  		FIG. 3. TRAIL-induced apoptosis in sensitized blastocysts. A) Blastocysts were recovered and cultured in vitro in the presence of medium alone, TRAIL, actinomycin D, or a combination of TRAIL and actinomycin D. The TUNEL assay was performed. Apoptotic nuclei are depicted in red; all nuclei are stained blue with a nuclear dye. The data are representative of three independent experiments with a minimum of seven embryos per treatment group for each experiment. B) Percentage of TUNEL-positive nuclei demonstrating DNA fragmentation per total embryonic nuclei. All data are expressed as the mean ± SEM. The TRAIL plus actinomycin D-treatment group was determined to be statistically different (denoted by *, P < 0.01) from all other treatments via an ad hoc ANOVA coupled with a Bonferroni/Dunn test


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Exposure of preimplantation embryos to toxins for short periods of time during the preimplantation period can result in the formation of neural tube defects, limb abnormalities, and abdominal wall malformations. Similar malformations are also seen among infants of women with diabetes [31– 36]. We and others have previously shown that maternal hyperglycemia or high concentration of glucose in vitro triggers apoptosis during the preimplantation period [2, 3]. Studies have also suggested that this glucose-induced cell death during the preimplantation period might be involved in the development of diabetes-associated embryopathy [37, 38]. Gaining a greater understanding of the mechanisms by which embryos undergo programmed cell death may prove to be important for elucidating how certain cellular stresses induce apoptosis in early mammalian embryos and how, in turn, this may influence later embryo development.

Programmed cell death in preimplantation embryos is not well defined; however, the molecular mechanisms underlying this process are beginning to become more clear. The preimplantation embryo appears to be vulnerable to cell death at two definitive periods: during early cleavage, and at the blastocyst stage [39–44]. Nuclear fragmentation in early rodent and human embryos has been associated with the activation of programmed cell death and appears to occur just before embryonic genome activation, a point at which the embryo switches from utilizing maternally inherited to embryonically synthesized proteins [42, 44, 45]. Apoptosis, which normally occurs at a low rate during the blastocyst stage, is thought to deplete the inner cell mass of cells that maintain the ability to differentiate into trophectoderm [46]. It has been postulated that apoptosis in early embryos may be a way to eliminate defective progeny [44].

In the present study, we have identified the presence of TRAIL and MK, a death-inducing ligand and its receptor, throughout murine preimplantation embryo development. These proteins are located mainly at the plasma membrane of the individual blastomeres from the 1-cell through the early morula stage of embryo development. At the blastocyst stage, 2-cell lineages arise from the totipotent blastomeres. The inner cell mass gives rise to the embryo proper; the trophectoderm cells develop into the placenta. The predominant apical staining pattern seen on blastocysts indicates that TRAIL and MK are located on the trophectoderm cells. Because MK must be located at the plasma membrane to transduce its apoptotic signal, only trophectoderm cells and, thus, a fraction of cells within the blastocyst likely are susceptible to TRAIL-induced apoptosis. The significance of the cytoplasmic staining in the trophectoderm cells is not known, but it is possible that under some conditions of stress, more TRAIL and MK could move to the apical plasma membrane. Developmentally, the location of the proteins is logical. Trophectoderm cells will form the placenta and, thus, will have the greatest interaction with the maternal environment; therefore, these proteins should be expressed at the plasma membrane in this cell lineage. Perhaps TRAIL plays a role at the maternal-fetal interface in apoptosis induction as well as in the generation of immune tolerance during pregnancy.

Intriguingly, TRAIL induces apoptosis in a number of neoplastic cell lines, but it does not appear to be toxic to normal cells [8, 9, 47, 48]. The mechanism of TRAIL resistance in normal cells is controversial; however, current theories include 1) the presence of decoy receptors on the surface of normal cells and 2) the presence of cell death-inhibitor proteins, such as c-FLIP (cellular FLICE-like inhibitory protein) [7, 26, 49–55]. In addition, the relative levels of caspases and their inhibitors as well as other environmental factors may affect TRAIL sensitivity in cells.

Similarly, normal blastocysts are resistant to TRAIL-induced apoptosis. Only when the embryo is sensitized via actinomycin D, a nucleic acid-synthesis inhibitor, is it rendered susceptible to TRAIL-induced apoptosis. The combination of TRAIL with other cytotoxic agents, such as cycloheximide and radiation as well as actinomycin D, have been shown to sensitize cells to TRAIL-induced apoptosis [26, 29, 56, 57]. In some model systems, the mechanism of TRAIL resistance involves the cellular inhibitor of apoptosis c-FLIP. High levels of c-FLIP expression correlate with TRAIL resistance in certain cancer cell lines [26, 54]. Actinomycin D has been shown to decrease the levels of c-FLIP protein, thus rendering melanoma cells sensitive to TRAIL-induced cell death [26]. In fact, c-FLIP is expressed in normal murine blastocysts and, therefore, may confer resistance to TRAIL-induced cell death at this stage of preimplantation embryo development (data not shown).

In addition to inhibitors of transcription, certain cellular stresses have been shown to sensitize cells to death receptor-induced apoptosis. Hyperglycemia has been shown to induce apoptosis in diverse cell systems. Both intrinsic and extrinsic cell death pathways have been implicated in diabetes-induced programmed cell death. Glucose has been shown to enhance death receptor activation in several systems. Hypoxia-induced apoptosis mediated by the extrinsic pathway is enhanced by glucose uptake and metabolism [58]. Moreover, glucose can specifically regulate TRAIL-induced cytotoxicity. Glucose deprivation was shown to enhance TRAIL-induced apoptosis in cancer cells [59]. Thus, cellular stresses have been shown to sensitize cells to apoptosis induced by the extrinsic cell death pathway.

Studies have shown that death receptors other than MK and their corresponding ligands are present on mammalian preimplantation embryos. Both Fas and Fas ligand (FasL), which are important in the regulation of immune tolerance, are expressed early in embryo development. Fas mRNA is expressed only in late 2-cell rat embryos, whereas FasL mRNA is expressed in oocytes, 1-cell, and late 2-cell rat embryos [60]. Fas and FasL were not detected in rat blastocysts; however, a separate report demonstrated the presence of Fas in mouse embryonic stem cells, where it was shown to mediate apoptosis [61]. To date, no evidence demonstrates Fas-mediated apoptosis in intact blastocysts; thus, this protein may not be involved in apoptosis induction at this stage of development. FasL has been detected in the trophectoderm of both human and mouse blastocysts and on the surface of trophoblasts, whereas Fas is detected on endometrial epithelial cells, suggesting that this receptor-ligand system may be important in blastocyst implantation [62, 63].

TNF{alpha} is a proinflammatory cytokine that is produced in the uterus of rodents during the window of implantation [64]. TNF{alpha} receptors are expressed in blastocysts, embryonic stem cells, and trophoblast cells [65–68]. TNF{alpha} has been shown to decrease cellular proliferation and glucose uptake in blastocysts and been reported to increase the number of TUNEL-positive nuclei in these embryos [3, 69]. However, antisense inhibition of TNF p60 receptor (TNFRp60) expression did not reverse the high frequency of dead cells seen in rat blastocysts exposed to media conditioned with diabetic uterine cells and, thereby, enriched in TNF{alpha} [70]. Antisense treatment did not result in a complete block in TNFRp60 expression, but the role of TNF{alpha} and its corresponding death receptor in blastocyst programmed cell death is unclear.

The data presented herein are the first to demonstrate the presence and function of TRAIL and MK, a death-inducing ligand and its receptor, on mammalian preimplantation embryos. We speculate that this receptor-ligand pair may be activated in certain disease states, such as maternal hyperglycemia, resulting in the induction of apoptosis via the extrinsic cell death pathway in preimplantation embryos. This increase in the level of apoptosis within the blastocyst combined with the apoptosis induced by hyperglycemia via activation of the intrinsic pathway could then lead to the increased incidences of malformations and miscarriages that are seen later in embryonic development.


   ACKNOWLEDGMENTS
 
We would like to thank J. Michael White (Washington University) for his expertise and assistance with embryo manipulation and culture.


   FOOTNOTES
 
1 Supported by a Lalor Foundation Fellowship to J.K.R. and by HD38061-01 from the National Institutes of Health to K.H.M. Back

2 Correspondence: Kelle H. Moley, 4911 Barnes-Jewish Hospital Plaza, St. Louis, MO 63110. FAX: 314 747 4150; moleyk{at}msnotes.wustl.edu Back

Received: 23 December 2003.

First decision: 11 January 2004.

Accepted: 29 April 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Baehrecke EH. How death shapes life during development. Nat Rev Mol Cell Biol 2002 3:779-787[CrossRef][Medline]
  2. Moley KH, Chi MM-Y, Knudson CM, Korsmeyer SJ, Mueckler MM. Hyperglycemia induces apoptosis in preimplantation embryos via cell death effector pathways. Nat Med 1998 12:1421-1424
  3. Pampfer S, Vanderheyden I, McCracken JE, Vesela J, De Hertogh R. Increased cell death in rat blastocysts exposed to maternal diabetes in utero and to high glucose or tumor necrosis factor {alpha} in vitro. Development 1997 124:4827-4836[Abstract]
  4. Moley KH. Hyperglycemia and apoptosis: mechanisms for congenital malformations and pregnancy loss in diabetic women. Trends Endocrinol Metab 2001 12:78-82[CrossRef][Medline]
  5. Roth W, Reed JC. Apoptosis and cancer: when BAX is TRAILing away. Nat Med 2002 8:216-218[CrossRef][Medline]
  6. Gupta S. Molecular signaling in death receptor and mitochondrial pathways of apoptosis. Int J Oncol 2003 22:15-20[Medline]
  7. Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science 1998 281:1305-1308[Abstract/Free Full Text]
  8. Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, Sutherland GR, Smith TD, Rauch C, Smith CA. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995 3:673-682[CrossRef][Medline]
  9. Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A. Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 1996 271:12687-12690[Abstract/Free Full Text]
  10. Wu G, Burns T, Zhan Y, Alnemri E, El-Deiry W. Molecular cloning and functional analysis of the mouse homologue of the KILLER/DR5 tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor. Cancer Res 1999 59:2770-2775[Abstract/Free Full Text]
  11. MacFarlane M. TRAIL-induced signaling and apoptosis. Toxicol Lett 2003 139:89-97[CrossRef][Medline]
  12. Schneider P, Olson D, Tardivel A, Browning B, Lugovskoy A, Gong D, Dobles M, Hertig S, Hofmann K, Van Vlijmen H, Hsu YM, Burkly LC, Tschopp J, Zheng TS. Identification of a new murine tumor necrosis factor receptor locus that contains two novel murine receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). J Biol Chem 2003 278:5444-5454[Abstract/Free Full Text]
  13. Bodmer JL, Holler N, Reynard S, Vinciguerra P, Schneider P, Juo P, Blenis J, Tschopp J. TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat Cell Biol 2000 2:241-243[CrossRef][Medline]
  14. Sprick MR, Weigand MA, Rieser E, Rauch CT, Juo P, Blenis J, Krammer PH, Walczak H. FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2. Immunity 2000 12:599-609[CrossRef][Medline]
  15. Kischkel FC, Lawrence DA, Chuntharapai A, Schow P, Kim KJ, Ashkenazi A. Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity 2000 12:611-620[CrossRef][Medline]
  16. Brojatsch J, Naughton J, Rolls MM, Zingler K, Young JA. CAR1, a TNFR-related protein, is a cellular receptor for cytopathic avian leukosis-sarcoma viruses and mediates apoptosis. Cell 1996 87:845-855[CrossRef][Medline]
  17. Bridgham JT, Johnson AL. Avian TVB (DR5-like) death receptor expression in hen ovarian follicles. Biochem Biophys Res Commun 2002 291:226-232[CrossRef][Medline]
  18. Manabe M, Myoumoto A, Tajima C, Fukumoto M, Nakayama M, Uchio K, Yamaguchi M, Miyamoto H. Immunochemical characteristics of a novel cell death receptor and a decoy receptor on granulosa cells of porcine ovarian follicles. Cytotechnology 2000 33:189-201[CrossRef]
  19. Manabe M, Myoumoto A, Tajima C, Nakayama M, Yamaguchi M, Yamada-Uchio K, Miyamoto H. Monoclonal antibodies recognize a novel cell death receptor and a decoy receptor on granulosa cells of porcine ovarian follicles. Journal of Mammalian Ova Research 2001 18:1-13
  20. Wada S, Manabe N, Nakayama M, Inou N, Matsui T, Miyamoto H. TRAIL-decoy receptor 1 plays inhibitory role in apoptosis of granulosa cells from pig ovarian follicles. J Vet Med Sci 2002 64:435-439[CrossRef][Medline]
  21. Phillips TA, Ni J, Pan G, Ruben SM, Wei YF, Pace JL, Hunt JS. TRAIL (Apo-2L) and TRAIL receptors in human placentas: implications for immune privilege. J Immunol 1999 162:6053-6059[Abstract/Free Full Text]
  22. Phillips TA, Ni J, Hunt JS. Death-inducing tumor necrosis factor (TNF) superfamily ligands and receptors are transcribed in human placentae, cytotrophoblasts, placental macrophages and placental cell lines. Placenta 2001 22:663-672[CrossRef][Medline]
  23. Moley KH, Chi M, Mueckler M. Maternal hyperglycemia alters glucose transport and utilization in mouse preimplantation embryos. Am J Physiol 1998 275:E38-E47
  24. Hogan A, Heyner S, Charron MJ, Copeland NG, Gilbert DJ, Jenkins MA, Thorens B, Schultz GA. Glucose transporter gene expression in early mouse embryos. Development 1991 113:363-372[Abstract]
  25. Aghayan M, Rao LV, Smith RM, Jarett L, Charron MJ, Thorens B, Heyner S. Developmental expression and cellular localization of glucose transporter molecules during mouse preimplantation development. Development 1992 115:305-312[Abstract]
  26. Griffith TS, Chin WA, Jackson GC, Lynch DH, Kubin MZ. Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells. J Immunol 1998 161:2833-2840[Abstract/Free Full Text]
  27. Matsuzaki H, Schmied BM, Ulrich A, Standop J, Schneider MB, Batra SK, Picha KS, Pour PM. Combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and actinomycin D induces apoptosis even in TRAIL-resistant human pancreatic cancer cells. Clin Cancer Res 2001 7:407-414[Abstract/Free Full Text]
  28. Olsson A, Diaz T, Aguilar-Santelises M, Osterborg A, Celsing F, Jondal M, Osorio LM. Sensitization to TRAIL-induced apoptosis and modulation of FLICE-inhibitory protein in B chronic lymphocytic leukemia by actinomycin D. Leukemia 2001 15:1868-1877[Medline]
  29. Zisman A, Ng CP, Pantuck AJ, Bonavida B, Belldegrun AS. Actinomycin D and gemcitabine synergistically sensitize androgen-independent prostate cancer cells to Apo2L/TRAIL-mediated apoptosis. J Immunother 2001 24:459-471
  30. Hernandez A, Wang QD, Schwartz SA, Evers BM. Sensitization of human colon cancer cells to TRAIL-mediated apoptosis. J Gastrointest Surg 2001 5:56-65[CrossRef][Medline]
  31. Generoso WM, Rutledge JC, Cain KT, Hughes LA, Braden PW. Exposure of female mice to ethylene oxide within hours after mating leads to fetal malformation and death. Mutat Res 1987 176:269-274[Medline]
  32. Greene MF, Hare JW, Cloherty JP, Benacerraf BR, Soeldner JS. First-trimester hemoglobin A1 and risk for major malformation and spontaneous abortion in diabetic pregnancy. Teratology 1989 39:225-231[CrossRef][Medline]
  33. Kimmel CA, Generoso WM, Thomas RD, Bakshi KS. A new frontier in understanding the mechanisms of developmental abnormalities. Toxicol Appl Pharmacol 1993 119:159-165[CrossRef][Medline]
  34. Hawthorne G, Robson S, Ryall EA, Sen D, Roberts SH, Ward Platt MP. Prospective population based survey of outcome of pregnancy in diabetic women: results of the Northern Diabetic Pregnancy Audit, 1994 [see comments]. BMJ 1997 315:279-281[Abstract/Free Full Text]
  35. Polifka JE, Rutledge JC, Kimmel GL, Dellarco V, Generoso WM. Exposure to ethylene oxide during the early zygotic period induces skeletal anomalies in mouse fetuses. Teratology 1996 53:1-9[CrossRef][Medline]
  36. Pregnancy outcomes in the Diabetes Control and Complications Trial [see comments]. Am J Obstet Gynecol 1996 174:1343-1353[Medline]
  37. Wuu YD, Pampfer S, Becquet P, Vanderheyden I, Lee KH, De Hertogh R. Tumor necrosis factor {alpha} decreases the viability of mouse blastocysts in vitro and in vivo. Biol Reprod 1999 60:479-483[Abstract/Free Full Text]
  38. Chi MM, Pingsterhaus J, Carayannopoulos M, Moley KH. Decreased glucose transporter expression triggers BAX-dependent apoptosis in the murine blastocyst. J Biol Chem 2000 275:40252-40257[Abstract/Free Full Text]
  39. Mohr LR, Trounson AO. Comparative ultrastructure of hatched human, mouse and bovine blastocysts. J Reprod Fertil 1982 66:499-504
  40. Handyside A, Hunter S. Cell division and death in the mouse blastocyst before implantation. Roux's Arch Dev Biol 1986 195:519-525[CrossRef]
  41. Jurisicova A, Varmuza S, Casper RF. Involvement of programmed cell death in preimplantation embryo demise. Hum Reprod Update 1995 1:558-566[Abstract/Free Full Text]
  42. Jurisicova A, Varmuza S, Casper RF. Programmed cell death and human embryo fragmentation. Mol Hum Reprod 1996 2:93-98[Abstract/Free Full Text]
  43. Brison DR, Schultz RM. Apoptosis during mouse blastocyst formation: evidence for a role for survival factors including transforming growth factor {alpha}. Biol Reprod 1997 56:1088-1096[Abstract]
  44. Jurisicova A, Latham KE, Casper RF, Varmuza SL. Expression and regulation of genes associated with cell death during murine preimplantation embryo development. Mol Reprod Dev 1998 51:243-253[CrossRef][Medline]
  45. Erenus M, Zouves C, Rajamahendran P, Leung S, Fluker M, Gomel V. The effect of embryo quality on subsequent pregnancy rates after in vitro fertilization. Fertil Steril 1991 56:707-710[Medline]
  46. Pierce GB, Lewellyn AL, Parchment RE. Mechanism of programmed cell death in the blastocyst. Proc Natl Acad Sci U S A 1989 86:3654-3658[Abstract/Free Full Text]
  47. Mariani SM, Matiba B, Armandola EA, Krammer PH. Interleukin 1ß-converting enzyme related proteases/caspases are involved in TRAIL-induced apoptosis of myeloma and leukemia cells. J Cell Biol 1997 137:221-229[Abstract/Free Full Text]
  48. Mariani SM, Krammer PH. Differential regulation of TRAIL and CD95 ligand in transformed cells of the T and B lymphocyte lineage. Eur J Immunol 1998 28:973-982[CrossRef][Medline]
  49. Gura T. How TRAIL kills cancer cells, but not normal cells. Science 1997 277:768[Free Full Text]
  50. Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 1997 277:815-818[Abstract/Free Full Text]
  51. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, Ramakrishnan L, Gray CL, Baker K, Wood WI, Goddard AD, Godowski P, Ashkenazi A. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 1997 277:818-821[Abstract/Free Full Text]
  52. Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, Steiner V, Bodmer JL, Schroter M, Burns K, Mattmann C, Rimoldi D, French LE, Tschopp J. Inhibition of death receptor signals by cellular FLIP. Nature 1997 388:190-195[CrossRef][Medline]
  53. Mongkolsapaya J, Cowper AE, Xu XN, Morris G, McMichael AJ, Bell JI, Screaton GR. Lymphocyte inhibitor of TRAIL (TNF-related apoptosis-inducing ligand): a new receptor protecting lymphocytes from the death ligand TRAIL. J Immunol 1998 160:3-6[Abstract/Free Full Text]
  54. Leverkus M, Neumann M, Mengling T, Rauch CT, Brocker EB, Krammer PH, Walczak H. Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes. Cancer Res 2000 60:553-559[Abstract/Free Full Text]
  55. Walczak H, Krammer PH. The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems. Exp Cell Res 2000 256:58-66[CrossRef][Medline]
  56. Ahmad M, Shi Y. TRAIL-induced apoptosis of thyroid cancer cells: potential for therapeutic intervention. Oncogene 2000 19:3363-3371[CrossRef][Medline]
  57. Nesterov A, Ivashchenko Y, Kraft AS. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in normal prostate epithelial cells. Oncogene 2002 21:1135-1140[CrossRef][Medline]
  58. Malhotra R, Lin Z, Vincenz C, Brosius FC III. Hypoxia induces apoptosis via two independent pathways in Jurkat cells: differential regulation by glucose. Am J Physiol 2001 281:C1596-C1603
  59. Nam SY, Amoscato AA, Lee YJ. Low glucose-enhanced TRAIL cytotoxicity is mediated through the ceramide-Akt-FLIP pathway. Oncogene 2002 21:337-346[CrossRef][Medline]
  60. Kawamura K, Fukuda J, Kodama H, Kumagai J, Kumagai A, Tanaka T. Expression of Fas and Fas ligand mRNA in rat and human preimplantation embryos. Mol Hum Reprod 2001 7:431-436[Abstract/Free Full Text]
  61. Zou GM, Reznikoff-Etievant MF, Leon A, Verge V, Hirsch F, Milliez J. Fas-mediated apoptosis of mouse embryo stem cells: its role during embryonic development. Am J Reprod Immunol 2000 43:240-248
  62. Bamberger AM, Schulte HM, Thuneke I, Erdmann I, Bamberger CM, Asa SL. Expression of the apoptosis-inducing Fas ligand (FasL) in human first- and third-trimester placenta and choriocarcinoma cells. J Clin Endocrinol Metab 1997 82:3173-3175[Abstract/Free Full Text]
  63. Galan A, O'Connor JE, Valbuena D, Herrer R, Remohi J, Pampfer S, Pellicer A, Simon C. The human blastocyst regulates endometrial epithelial apoptosis in embryonic adhesion. Biol Reprod 2000 63:430-439[Abstract/Free Full Text]
  64. Yelavarthi KK, Chen HL, Yang YP, Cowley BD Jr, Fishback JL, Hunt JS. Tumor necrosis factor {alpha} mRNA and protein in rat uterine and placental cells. J Immunol 1991 146:3840-3848[Abstract]
  65. Pampfer S, Wuu YD, Vanderheyden I, De Hertogh R. Expression of tumor necrosis factor {alpha} (TNF{alpha}) receptors and selective effect of TNF{alpha} on the inner cell mass in mouse blastocysts. Endocrinology 1994 134:206-212[Abstract]
  66. Kohchi C, Tanabe Y, Noguchi K, Mizuno D, Soma G. Induction of differentiation in embryonic stem cells by 26-kD membrane-bound tumor necrosis factor (TNF) and 17-kD free TNF. In Vivo 1996 10:19-27[Medline]
  67. Ben-Yair E, Less A, Lev S, Ben-Yehoshua L, Tartakovsky B. Tumor necrosis factor {alpha} binding to human and mouse trophoblast. Cytokine 1997 9:830-836[CrossRef][Medline]
  68. Wuu YD, Pampfer S, Vanderheyden I, Lee KH, De Hertogh R. Impact of tumor necrosis factor {alpha} on mouse embryonic stem cells. Biol Reprod 1998 58:1416-1424[Abstract/Free Full Text]
  69. Pampfer S, Moulaert B, Vanderheyden I, Wuu YD, De Hertogh R. Effect of tumor necrosis factor {alpha} on rat blastocyst growth and glucose metabolism. J Reprod Fertil 1994 101:199-206
  70. Pampfer S, Vanderheyden I, Vesela J, De Hertogh R. Neutralization of tumor necrosis factor {alpha} (TNF{alpha}) action on cell proliferation in rat blastocysts by antisense oligodeoxyribonucleotides directed against TNF{alpha} p60 receptor. Biol Reprod 1995 52:1316-1326[Abstract]



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C. Rieger, R. Poppino, R. Sheridan, K. Moley, R. Mitra, and D. Gottlieb
Polony analysis of gene expression in ES cells and blastocysts
Nucleic Acids Res., December 10, 2007; (2007) gkm1076v1.
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