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Regular Article |
a Division Maternal-Fetal Medicine,
b Reproductive Biology Unit, and
c Division of Reproductive Medicine, Department of Obstetrics and Gynecology, University of Ottawa, and the Ottawa Hospital Loeb Research Institute, Ottawa Hospital, Ottawa, Ontario, Canada K1H 8L6
ABSTRACT
In this study, we have examined the expression and potential role of X-linked inhibitor of apoptosis protein (XIAP), Fas, and Fas ligand (FasL) in the regulation of apoptosis throughout placental development. Protein expression was determined by Western blot analysis and immunohistochemistry, whereas apoptotic cell death was assessed by DNA fragmentation analysis and TUNEL. The XIAP was present in trophoblast throughout placental development, but its content significantly decreased during late pregnancy, when apoptosis was maximal. The FasL content was low during early placental development but increased coincidentally to the decrease in XIAP during the third trimester. Our data also suggest that placental apoptosis is the culmination of the relative expression of these cell-death and -survival proteins, a phenomenon that is cell type-specific and dependent on cytodifferentiation and the stage of placental development. Moreover, the induction of syncytiotrophoblast apoptosis may involve the concomitant up-regulation of FasL for Fas activation and the removal of downstream inhibition of the apoptotic cascade by XIAP.
apoptosis, developmental biology, placenta, pregnancy, trophoblast
INTRODUCTION
Fetal growth is dependent upon a variety of factors, which may be genetic, environmental, metabolic, hormonal, and placental in nature. The placenta influences fetal growth by at least three mechanisms: changes in its own metabolism, its capacity to transport oxygen and nutrients, and its functional size. Indeed, placental growth parallels fetal growth until near term. In addition, restricted placental growth, such as that seen in pre-eclampsia, is associated with decreased fetal growth [1].
Elucidation of the regulatory mechanisms of placental growth might provide important clues in our understanding of the controls of fetal growth. Placental growth is complex and influenced by many factors, and it is dependent upon a delicate balance between cell proliferation, differentiation, and death. Apoptosis is a programmed, physiologic form of cell death important in the control of cell population. It is characterized by nuclear condensation and fragmentation, membrane blebbing, and condensation of cytoplasm with preservation of organelle integrity [2], and it is involved in both physiologic and pathologic situations, such as vertebrate development, sloughing of endometrium during menses, ovarian follicular atresia, and various forms of cancer and immune disorders [3, 4]. To date, little information exists on the incidence and potential role of apoptosis throughout placental development. Apoptosis has been observed in the placenta throughout gestation, with increased frequency during the third trimester [5] as well as in placentae of growth-restricted fetuses [6].
Activation of the apoptotic cascade results from a complex interaction of molecular events, including cross-linking Fas (CD 95), a cell surface-receptor protein of the tumor necrosis factor (TNF)-receptor family, to its ligand (FasL) [7]. Two forms of FasL exist: a membrane-associated form (mFasL), and a soluble form (sFasL) that results from the cleavage of mFasL by metalloproteinases. Although both forms can induce apoptosis, the relative importance of the two ligands is not clear [8]. Fas also exists in membrane-bound and soluble forms, with the latter being the product of alternate splicing, which eliminates the transmembrane domain and may modulate the role of the full-length Fas protein. Although both Fas and FasL are expressed in trophoblasts throughout gestation, their role in apoptosis has recently been questioned [9].
The inhibitors of apoptosis proteins (IAPs) are a group of intracellular survival proteins first identified in baculoviruses, the function of which was to keep the host cells alive while the virus continued to replicate [10, 11]. Five mammalian IAPs have been identified: neuronal apoptosis-inhibitory protein (NAIP), human inhibitor of apoptosis protein (HIAP)-1, HIAP-2, X-linked inhibitor of apoptosis protein (XIAP), and survivin [12–16]. Of these, XIAP appears to be important in the control of apoptosis through modulation of caspase activation and activity, key events in the apoptotic cascade. It attenuates Fas-mediated apoptosis by inhibiting the activity of caspase-3, a cell-death protease important in the downstream signaling of apoptosis [17]. To our knowledge, the localization and potential role of this survival protein in the placenta have not been investigated.
In the present studies, we have examined the expression and possible role of XIAP and the Fas/FasL system in the regulation of apoptosis during placental growth. We have assessed the relative expression of these proteins and the cell type involved in relation to apoptotic cell death in the human placenta during each trimester by immunohistochemistry and TUNEL, respectively. Our findings suggest that placental apoptosis is the culmination of the relative expression of these cell-death and -survival proteins, a phenomenon that is cell type-specific and dependent upon cytodifferentiation and stage of placental development.
MATERIALS AND METHODS
Sample Collection
Placental tissues were obtained from 25 first-trimester and 8 second-trimester pregnancies immediately after therapeutic termination. In addition, placental tissues from 26 third-trimester pregnancies were also obtained immediately at birth. All first- and second-trimester pregnancies were dated with ultrasound, whereas third-trimester pregnancies were dated with either early ultrasound or sure last menstrual period. No cases of intrauterine fetal death, congenital or chromosomal anomalies, or multiple gestations were included. No fetal growth anomalies (i.e., all birth weights were between the 10th and 90th percentiles) or pregnancy complications were present. In particular, no history of smoking or drug use was involved. This study was approved by the Research Ethics Board of the Ottawa Hospital.
First- and second-trimester placental specimens were divided in two portions: one immediately frozen at -70°C, and another fixed in 4% (v/v) paraformaldehyde. The latter samples were subsequently embedded in paraffin, and 4–5-µm sections were mounted on charged slides. Placentae obtained from third-trimester pregnancies were sampled randomly over two to four areas. The specimens were then processed in the same manner as those collected during the first and second trimesters.
Identification and Quantification of Apoptosis
DNA ladder analysis
Apoptotic cell death was assessed on the basis of DNA fragmentation and confirmed with the visualization of discrete DNA fragments of 185-base pair multiples on agarose gel electrophoresis. The DNA was extracted from each specimen using the Qiagen Tissue Amp Kit (Qiagen, Chatsworth, CA) and end-labeled by incubating with terminal end-transferase (Roche Molecular Biochemicals, Laval, PQ, Canada) and 
-32P-2',3'-dideoxyadenosine 5' triphosphate (ddATP). Briefly, a 500-ng DNA sample was added to a mixture 5 µl of 5x terminal deoxynucleotidyl transferase (TdT) buffer, 2.5 µl of 10x CoCl2, 0.5 µl of TdT enzyme, and 0.5 µl of 10 mCi/ml of 32P-ddATP and AE buffer (Quiagen) to total volume of 25 µl, then incubated at 37°C for 60 min. Unincorporated nucleotides were removed using the Qiagen nucleotide removal kit (Qiagen), and the samples were subsequently resolved by 1.8% agarose. The dried gel was then exposed to a BioRad (Hercules, CA) phosphoimager, and low molecular weight DNA (<6 kilobase pairs) and genomic DNA were densitometrically quantified. The gel was then exposed to radiographic film at -80°C. To correct for possible uneven gel loading, the ratio of low molecular weight DNA (representing apoptosis) to genomic DNA was calculated for each sample, and the means of ratios in each trimester were compared. Examination of multiple samples for each placenta (e.g., third-trimester specimens) indicated that the variability in apoptosis in different areas within the same placenta was less than 20%. The intraobserver variability as determined by performing two separate DNA ladder analyses on the same sample was approximately 5%. The intraplacental variability justified the need for examining several areas of a large number of placentae, especially during the third trimester, and use of the means of these results for comparisons.
TUNEL analysis The TUNEL analysis was performed to identify the cell type(s) undergoing apoptosis using the In Situ Cell Death Detection Kit POD (Roche). Mounted sections were stained with hematoxylin-and-eosin (H&E) for routine histology and examined under light microscopy. Subsequently, adjacent sections were treated (1 min) with microwave in 0.01 M citrate buffer (pH 6.0). They were then washed with PBS and incubated with terminal transferase in the presence of fluorescein isothiocyanate-conjugated 2' deoxyuridine 5' triphosphate. Signal was converted with anti-fluorescein antibody conjugated with horse radish peroxidase (HRP) and 3,3'-diaminobenzidine (DAB) substrate. Positive controls were generated by incubation with DNase I (1 µg/ml before labeling). Slides were then photographed under light microscopy, and positive cells were identified by two blinded investigators separately. The incidence of apoptosis was scored in terms of the well-described apoptotic index [18–20]. Briefly, slides were examined at 400x magnification with an oil immersion lens. Approximately five fields were selected at random, and a total of 1000 nuclei were counted, avoiding the marginal areas of the stained specimen. The number of apoptotic trophoblast nuclei was expressed as a percentage of the total trophoblast nuclei counted (i.e., apoptotic index).
Western Blot Analysis
Placental tissues (50–100 mg) were homogenized in RIPA (1x PBS, 1% Nonidet P-40 [Sigma, Oakville, ON, Canada], 0.5% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml of aprotinin, and 10 µg/ml of leupeptin). The homogenate were sonicated (10 sec) and then centrifuged (14 000 x g, 4°C, 20 min). The protein content of the supernatant was determined with the BioRad DC protein assay kit (BioRad). Aliquots of protein (50 µg) were resolved by SDS-PAGE and electrotransferred to nitrocellulose membranes, including one pooled sample as an internal control in each gel. The membranes were blocked (1 h, room temperature [RT]) in blotto (Tris-buffered saline [pH 7.6] with 0.05% Tween 20 [TBS-T] and 5% dehydrated nonfat milk). After washing in TBS-T (four times, 5 min each), the membranes were incubated (1 h, RT) with each antibody (Fas [C-20], FasL [C-178] for mFasL, FasL [C-20] for sFasL, proliferating cell nuclear antigen [PCNA; Santa Cruz Biotechnology Inc., Santa Cruz, CA], and XIAP [ApoptoGen, Ottawa, ON, Canada]) diluted in blotto. The PCNA, a 36-kDa nuclear protein expressed in high abundance in the late G1 and S phases, has been used extensively as a marker of cell proliferation [21, 22]. Because tissue homeostasis depends on a balance between proliferation, differentiation, and death, PCNA content was examined to assess the contribution of cell proliferation in placental growth. After washing, the membranes were incubated with human IgG preabsorbed HRP-conjugated second antibody (1:2000) in blotto for another 1 h and washed again. Peroxidase activity was visualized with the enhanced chemiluminescence kit according to the manufacturer's instructions. The protein content was determined desitometrically and normalized with an internal control on the same membrane.
Specificity of the Western blot for each protein was confirmed in the present studies. Specifically, preincubating of the anti-FasL (C-20) with C-terminal, 20-amino acid peptide completely blocked 26-kDa soluble FasL. Also, the 55-kDa XIAP band was blocked by preabsorption of anti-XIAP with recombinant XIAP protein. In addition, the commonly available HRP-conjugated anti-mouse or anti-rabbit IgG second antibody (BioRad) exhibits extensive nonspecific signals, but a similar second antibody, which was been pre-absorbed with human IgG, was also used in the present studies. The blots obtained with this latter antibody exhibited no bands other than that of the primary antibody. Moreover, internal controls were included when multiple gels were run to semiquantify the protein levels by Western blot analysis. Briefly, an aliquot of an internal control was applied to each gel, and the intensity of the signal (i.e., band) in the samples was normalized with that of the internal control to remove interassay variation (i.e., the ratios of the protein level of each sample relative to the level of the internal control were assessed).
Immunohistochemistry
Cubes of placenta fixed in 10% neutral buffered formalin were dehydrated through a graded series of ethanol and embedded in paraffin. Serial sections (4–5 µm) were deparaffinized using xylene and a graded series of ethanol. For FasL and XIAP immunostaining, slides were heated by microwave for 15 min in 0.01 M citrate buffer (pH 6.0). They were then immunostained with the Dako LSAB Kit (Dako Corporation, Mississauga, ON, Canada). Briefly, the sections were incubated for 10 min in PBS with 3% H2O2 solution and, after blocking, with monoclonal Fas antibody clone 13 (1:25, 10 µg/ml), FasL (C-178, 1:50, 4 µg/ml), XIAP (1:50, 20 µg/ml), and PCNA (1:25, 4 µg/ml) for 30 min at RT. The samples were incubated with biotinylated second antibodies (30 min), then with HRP-conjugated streptavidin, and visualized with 3-amino-9 ethylcarbazole (AEC). The specimens were counterstained with H&E. To determine the extent of nonspecific immunostaining, primary antibodies were substituted with mouse or rabbit IgG (at the same concentrations and isotype as primary antibodies) for use as negative controls. The signals were negligible compared to those with the primary antibodies. A stain-destain-stain technique was used to assess the TUNEL signal and XIAP expression in same cells. Briefly, TUNEL was performed first as described above, except that AEC (instead of DAB) was used as a substrate. After TUNEL images were captured, TUNEL-labeled sections were destained with 1% HCl in 70% ethanol (1 min) to remove residual signals, and XIAP immunohistochemistry was then performed in the same section as described above. The TUNEL-labeling signals and XIAP immunoreactivity of the same cells were compared.
Statistics
Data were logarithmically transformed to remove heterogeneity of variance before analysis by one-way ANOVA. Differences between experimental groups were assessed by the Student-Newman-Keuls test. Statistical significance was inferred at P < 0.05.
RESULTS
Identification and Quantification of Proliferation and Apoptosis
A total of 59 placentae were examined in detail across gestation. Apoptosis as assessed by the presence of DNA ladders and TUNEL-positive cells was present in all specimens. To identify the different cell types undergoing apoptosis, we examined TUNEL-labeled sections from each specimen and, after H&E staining, confirmed morphologically for the presence of cellular shrinkage and nuclear chromatin condensation. Cytokeratin-8 immunostaining was performed to identify trophoblasts (data not shown). Apoptosis was evident in all cell types in the placenta throughout development, but the extent in each cell type varied between trimesters.
Figure 1 illustrates representative samples of immunostaining for TUNEL, Fas, FasL, and PCNA throughout pregnancy. During the first trimester, apoptotic cell death as identified by TUNEL positivity was evident mostly in cytotrophoblasts and stromal cells (Fig. 1); occasionally, syncytiotrophoblasts also displayed apoptotic features. The incidence of syncytiotrophoblast apoptosis increased significantly from the first to the third trimester (2.5% ± 0.7% vs. 11.0% ± 1.8%, P = 0.005). The incidence of apoptosis in stromal cells remained high but unchanged throughout development, whereas cytotrophoblasts were rarely TUNEL-positive by the third trimester. Placental apoptotic DNA fragmentation as determined by quantitation of DNA ladders was not significantly different throughout pregnancy, but cell proliferative responses as assessed by Western blot analysis of changes in PCNA contents decreased markedly by the third trimester (P < 0.05; Fig. 2). The PCNA-labeled cells were primarily cytotrophoblasts and were most evident during the first and second trimesters (Fig. 1).
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Fas/FasL Expression and Signaling
To study the possible involvement of the Fas/FasL system in placental apoptotic signaling, we examined the expression of Fas and FasL by immunohistochemistry (Fig. 1) and Western blot analysis (Fig. 3). Immunosignal for Fas was observed in syncytiotrophoblasts, cytotrophoblasts, and stromal cells during the first trimester (Fig. 1). Fas immunoreactivity was strongest during the first trimester in the cytotrophoblast layer, whereas most intense signals were evident in syncytiotrophoblasts during the second trimester and at term. Fas immunosignals were also observed in stromal cells throughout gestation. Western blot analysis of protein extracts from the same samples revealed a 45-kDa band characteristic of the membrane-associated Fas receptor (Fig. 3). Quantification of Fas content revealed no statistically significant changes throughout gestation.
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The FasL was consistently localized primarily in trophoblasts for all trimesters (Fig. 1). During the first trimester, FasL was present in both syncytiotrophoblasts and cytotrophoblasts, although a preponderance of syncytiotrophoblasts stained positively. Later during development, and especially during the third trimester, only syncytiotrophoblasts stained positively for the ligand. Because both mFasL and sFasL can induce apoptosis, we quantified both forms of the protein across gestation by Western blot analysis (Fig. 2). An increase in the levels of both mFasL and sFasL was noted from the first to third trimesters. This was especially evident for the sFasL, which was present in low abundance until the third trimester (P < 0.05). A smaller increase in mFasL content was noted during the second trimester and persisted till term (P > 0.05).
XIAP Expression
The XIAP was localized in placenta in a pattern similar to that noted for FasL expression (Fig. 4). Indeed, both elements of the trophoblast layer stained positively for XIAP during the first trimester, with the most intense signals noted in the syncytiotrophoblasts. With further development, the syncytiotrophoblasts were the predominant cell type exhibiting XIAP immunoreactivity. Seldom was XIAP observed in TUNEL-positive cells throughout gestation (Fig. 4). Quantification of placental XIAP protein content (55 kDa) by Western blot analysis revealed relatively high abundance of XIAP during the first two trimesters of pregnancy and markedly lower levels during the third (Fig. 3). The decrease in XIAP level is of interest, because XIAP was localized in the syncytiotrophoblasts, which increased in number at term, specifically when apoptosis was maximal.
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DISCUSSION
The physiological role of XIAP in placental physiology is not known. To our knowledge, our report represents the first demonstration of the localization of XIAP in the placenta and its expression during development. We have shown that this intracellular survival protein is present throughout gestation, and that its level decreases significantly with the progress of pregnancy. It is present in both cytotrophoblasts and syncytiotrophoblasts during the first and second trimesters, although more abundantly in the syncytial layer. In addition, XIAP appears to be acquired during differentiation of the cytotrophoblast to syncytiotrophoblast during the first trimester and to be sustained until the third trimester, at which time a significant decrease in XIAP content and increase in apoptosis are noted, suggesting that XIAP may play an important role in the regulation of placental apoptosis throughout development. This notion is consistent with the present observations that XIAP expression was high in TUNEL-negative placental cells and undetectable in dying (i.e., TUNEL-positive) ones. The pattern of XIAP expression, particularly during the first trimester, is similar to those of Bcl-2 and Mcl-1, two intracellular proteins of the Bcl-2 family involved in the inhibition of apoptosis [23], although the relative importance of these cell-survival proteins in the regulation of placental function remains to be determined.
Although the Fas/FasL system is widely reported to be important in the activation of the apoptotic cascade in a variety of physiological systems [24–26], its role in the regulation of placental apoptosis has been equivocal. In the present studies, we observed, as others have previously reported [27–29], the discordant localization of Fas and FasL throughout placental development as well as a general dissociation of their expression from the incidence of apoptosis. Indeed, Fas immunosignals were strongest in cytotrophoblasts during the first trimester, but FasL was mostly localized in syncytiotrophoblasts. Moreover, despite strong FasL immunosignals in the syncytial layer during the first trimester, very few cells displaying Fas immunoreactivity were apoptotic. Placental Fas levels did not vary significantly throughout development. The significance of these apparent discrepancies is not well understood, but the Fas/FasL system may be more important during the first trimester for the maintenance of immune privilege [30–32], particularly because the strategic localization of FasL on syncytiotrophoblasts (i.e., at the maternal-fetal interface) as demonstrated by the present findings may be important for activation of apoptosis of maternal immune cells [32]. In addition, our results also suggest that its role in the initiation of placental apoptosis is not elaborated until the late stage of development (i.e., third trimester). Indeed, results from the present studies show that the progression of pregnancy into the third trimester is associated with an increase in placental sFasL and mFasL levels, a marked decrease in XIAP content, and an increase in the incidence of apoptosis, particularly in the syncytial layer. Because XIAP is an endogenous inhibitor of caspase-3, a cell-death protease downstream on the TNFR1 and Fas pathways [17], it is conceivable that during the third trimester, syncytiotrophoblast apoptosis is more extensive as a result of a loss of apoptotic inhibition with the disappearance of XIAP and increased Fas activation because of an overall increase in total FasL contents.
It is well established that FasL is a membrane protein of the TNF family [33] and is also present in a number of cellular systems as a soluble protein (i.e., sFasL) as a result of posttranslational processing by metalloproteinases (MMPs) [34]. Recent studies in other cellular systems have suggested both a pro- and anti-apoptotic role, depending on the cell type involved [35–38]. This raises the possibility that sFasL may have a cell-specific role in either blocking apoptotic cell death (potentially by interfering with internalization of the Fas/FasL complex) or promoting it by being more accessible to its target site on the Fas receptor [35, 36]. Results from the present studies show that the onset of syncytiotrophoblast apoptosis during the third trimester is associated with a more significant increase in the placental contents of sFasL than of mFasL. These findings suggest that an increase in FasL expression and, possibly, cleavage and "solubilization" of the mFasL by MMPs may be important for syncytiotrophoblast Fas-receptor activation and the induction of apoptosis. In this context, it is of interest to note that syncytiotrophoblasts of human placentae exhibit intense immunoreactivity for MMP-7, which increases at term (unpublished results). In addition, because MMP-7 is capable of processing mFasL to form sFasL in vitro, increased expression of MMP-7 in the human placenta during the late stage of pregnancy may, in part, represent an important mechanism by which sFasL is increased and syncytiotrophoblast apoptosis is initiated, as demonstrated in our current studies.
One potential limitation of the present study relates to the timing of tissue collection for third-trimester placentae. Whereas collecting placental tissues after birth was unavoidable, it is conceivable that parturition may influence the process of apoptosis and, therefore, bias the current findings. At present, controversy exists in the literature as to whether labor may be associated with modulations in placental apoptosis [39], and this requires further investigation. However, observations from our own laboratory (unpublished results) suggest the contrary and are consistent with those in a previous publication by Thiet et al. [39].
Results from the present studies clearly demonstrate that placental cell fate (i.e., proliferation, survival, and death) is determined by a balance in the expression, processing, or actions of cell-death and -survival factors, which are dependent on placental development and cytodifferentiation. We have assessed the expression of XIAP, FasL, and Fas in the context of their possible role in the regulation of placental apoptosis. We have quantified apoptosis throughout development, using both DNA ladders and TUNEL-labeling together with cellular morphological assessment of TUNEL-positive cells, because concerns have been expressed regarding possible labeling of ischemic or necrotic cells by the TUNEL method [40]. Whereas TUNEL labeling allowed determination of the cell death index throughout gestation, DNA fragmentation studies were informative regarding the nature of cell death (i.e., apoptosis vs. necrosis) but less useful in determining the cell types involved. Using TUNEL labeling, we observed that the cell death index in syncytiotrophoblasts increased from 2.5% to 11% during gestation. This increase is in agreement with a previous report [23], although the ranges of this index vary widely [5, 20, 41–43]. Consistent with previous observations [20, 41], apoptosis was evident in both cytotrophoblasts and syncytiotrophoblasts, although it was more prominent among the latter cell type at term.
In summary, we have shown that XIAP is present in trophoblasts throughout placental development and decreases significantly during late pregnancy, when the incidence of apoptosis increases. Our findings support the hypothesis that this intracellular protein plays an important role in modulating apoptotic cell death in trophoblasts, thereby maintaining syncytial integrity during early pregnancy, and that XIAP down-regulation later during development may be important in the control of placental growth. The present studies also suggest that syncytiotrophoblast apoptosis may, in part, be under the control of FasL expression and/or processing, but that it occurs only when inhibition of the downstream apoptotic pathway by XIAP is absent.
ACKNOWLEDGMENTS
We wish to thank Dr. Roman Feigel, the house staff, and the nursing staff of the Ottawa Hospital-General and Civic Campuses for their assistance in collecting placental samples. We are also indebted to Drs. William Gibb and Carl Nimrod for reviewing this manuscript. We thank Dr. Eric LaCasse of ApoptoGen for providing the XIAP antibody used in the current studies.
FOOTNOTES
First decision: 27 October 2000.
1 Supported by a grant from the Physician Services, Inc. (PSI). 
2 Correspondence: Andrée Gruslin, Division Maternal-Fetal Medicine, Department of Obstetrics, Gynecology and Newborn Care, Room 8420, Ottawa Hospital-General Campus, 501 Smyth Road, Ottawa, ON, Canada K1H 8L6. FAX: 613 737 8470; agruslin{at}ottawahospital.on.ca
Accepted: November 28, 2000.
Received: October 2, 2000.
REFERENCES
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