Molecular Human Reproduction, Vol. 5, No. 3, 246-251,
March 1999
© 1999 European Society of Human Reproduction and Embryology
Inverse relationship between apoptosis and Bcl-2 expression in syncytiotrophoblast and fibrin-type fibrinoid in early gestation
1 Department of Obstetrics and Gynecology, Shinshu University School of Medicine, 3–1–1 Asahi, Matsumoto 390-8621 and 2 Department of Gynecology and Obstetrics, Kyoto University Faculty of Medicine, Kyoto 606-8507, Japan
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The purpose of this study was to assess the role of apoptosis and cell cycle arrest in the trophoblast during early gestation by determining the location of apoptotic cells and examining the expression of Bcl-2 and p21. Using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) method on human chorionic villi, a cluster of apoptotic nuclei was demonstrated in perivillous fibrin-type fibrinoid, but no apoptotic changes were identified in the syncytiotrophoblast or in other subtypes of the trophoblast. The syncytiotrophoblast was diffusely positive for Bcl-2, but fibrin-type fibrinoid was negative for Bcl-2. Hence, there was an inverse relationship between apoptosis and Bcl-2 expression in both fibrin-type fibrinoid and syncytiotrophoblast. Expression of p21 was present to some extent in the syncytiotrophoblast, but not in fibrin-type fibrinoid. These results suggest that Bcl-2 may play an important role in preventing apoptosis in the syncytiotrophoblast; this may be necessary to prevent any DNA degradation from being spread to other nuclei in a multinuclear cell like the syncytiotrophoblast.
apoptosis/Bcl-2/histochemistry/p21/syncytiotrophoblast
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Introduction |
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The trophoblast of early gestation is considered to consist of syncytiotrophoblast, cytotrophoblast, and intermediate trophoblast on the basis of their location and morphology (Mazur and Kurman, 1994
; Benirschke and Kaufman, 1995). In addition, trophoblast that comprises the basal feet of the anchoring villi represents the most proliferative zone of the extravillous trophoblast and is specifically designated as the `trophoblast of the cell columns' (Benirschke and Kaufman, 1995). The proliferative activity of these subtypes of the trophoblast varies considerably; both the trophoblast of the cell columns and cytotrophoblast possess an extremely high proportion of cycling cells, while syncytiotrophoblast and intermediate trophoblast show practically no proliferation (Wakuda and Yoshida, 1992; Ichikawa et al., 1998). The presence of these different levels of proliferative activity among the subtypes of the trophoblast implies that different mechanisms regulating cell proliferation are present in the various subtypes.
Apoptosis is a form of programmed cell death that is controlled at the gene level, and it plays important roles in embryonic development, in the maintenance of tissue homeostasis, and in the elimination of cells that have suffered serious DNA damage (Kerr et al., 1994; Hale et al., 1996). Histologically, cells falling into apoptosis show characteristic morphological features such as cell shrinkage, nuclear chromatin compaction, pyknosis, nuclear fragmentation, cytoplasmic condensation, and convolution of the nuclear and cell outlines (Kerr et al., 1994; Cidlowski et al., 1996). It has been reported that in the trophoblast, apoptotic cells are detectable mainly in syncytiotrophoblast, either by electron microscopy (Nelson, 1996) or by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labelling (TUNEL) method (Yasuda et al., 1995; Smith et al., 1997). The latter method was developed by Gavrieli et al. (1992) and demonstrates nuclear DNA fragmentation. However, the localization of apoptotic cells in the trophoblast is not very well understood, especially in relation to apoptosis-regulating substances.
Apoptosis is regulated by a number of molecules including members of the Bcl-2 (B cell lymphoma/leukaemia-2) family (Reed, 1997). Bcl-2 was first discovered in human follicular lymphoma and was regarded as a proto-oncogene (Tsujimoto et al., 1985). Later, it was reported that Bcl-2 has the function of inhibiting apoptosis (Vaux et al., 1988; Kroemer et al., 1997). In normal human tissues, Bcl-2 is expressed in cells that actively proliferate, such as endometrial glandular cells in the proliferative phase (Tao et al., 1997; Watanabe et al., 1997). Thus, Bcl-2 induces immortalization of cells and enables them to proliferate actively. It has been reported that Bcl-2 expression is diffusely present in syncytiotrophoblast (Sakuragi et al., 1994; Lea et al., 1997). However, as we previously reported, syncytiotrophoblast has an extremely low proliferative activity (Ichikawa et al., 1998), and is considered to represent a mitotically end-stage cell (Benirschke and Kaufman, 1995). The diffuse Bcl-2 expression in syncytiotrophoblast is difficult to understand, since syncytiotrophoblast has little or no ability to proliferate and the inhibitory effect on apoptosis exerted by Bcl-2 generally leads to cell proliferation through immortalization of the cells. Moreover, it has not been reported how apoptosis is associated with Bcl-2 expression in the trophoblast at the individual cell level.
Cell proliferation is negatively regulated by the induction of cell cycle arrest, as well as by apoptosis (Hunter, 1993). Cell cycle inhibitory molecules, including p53 and p21, are important negative regulators of cell proliferation; their effects balance those of the growth-promoting forces, e.g. cyclins and cyclin-dependent kinases (CDKs) (El-Deiry et al., 1993; Harper et al., 1993; Dulic et al., 1994). p21, a strong suppressor of CDKs, is induced by p53; it inhibits cell cycling and leads to cell growth arrest. We recently found a strong relationship between the number of apoptotic cells and the number of p21-positive cells in the glandular cells of the endometrium (Toki et al., 1998). As yet, it is not clear whether there is also a relationship between apoptosis and p21 in the trophoblast. The purpose of this study was to investigate the role of apoptosis and cell cycle arrest in the trophoblast during early gestation by demonstrating apoptotic cells, Bcl-2 expression, and p21 expression.
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Reagents
Villous and decidual tissues of the first trimester were collected from patients who underwent elective termination of pregnancy at 6–12 weeks normal gestation (n = 15) and from hysterectomy specimens (n = 2) (8–12 weeks gestation). None of them showed any signs of miscarriage. The tissues were used with the approval of the Ethical Committee of Shinshu University, Japan. For the histochemical study, tissues were fixed with 10% phosphate-buffered formalin and embedded in paraffin. For Western blotting, fresh villous and decidual tissues were separately sampled from five cases in which normal gestation had progressed for 7–9 weeks, and stored at –80°C until protein extraction.
Detection of apoptotic cells showing DNA fragmentation
TUNEL method was performed using a Mebstain Apoptosis Kit (MBL, Nagoya, Japan) to detect in-situ nuclear DNA fragmentation or apoptosis. Sections were deparaffinized, rehydrated, and treated with 40 µg/ml proteinase K (Boehringer Mannheim, Mannheim, Germany) for 30 min at 37°C. After washing, they were incubated with TdT solution containing TdT and biotin-conjugated dUTP for 60 min at 37°C, and then with TB buffer (300 mM sodium chloride, 30 mM sodium citrate) for 15 min. They were washed again, treated with blocking solution, and incubated first with peroxidase-conjugated streptavidin (Nichirei) for 20 min, then with diaminobenzidine (DAB) for 5 min. An adjacent serial section pretreated with DNase 1 (Boehringer Mannheim) was used as a positive control. Sections of a human colon polyp (known to contain a number of apoptotic cells) were used as a positive tissue control. Negative control sections were incubated with TdT solution that did not contain TdT.
Immunohistochemistry
Immunohistochemical staining was performed on paraffin sections using the streptavidin–biotin–peroxidase complex method by means of a Histofine SAB-PO kit (Nichirei). The primary monoclonal antibodies used were Bcl-2 oncoprotein (Dakopatts, Glastrup, Denmark) and p21 (Pharmingen, San Diego, CA, USA). Sections were deparaffinized, rehydrated, and boiled in 0.01 M citrate buffer (pH 6.0) for 15 min in a microwave oven. After endogenous peroxidase activity had been blocked, they were incubated with normal rabbit serum to reduce non-specific binding. They were then incubated with the primary antibodies or normal mouse serum (negative control) at 4°C overnight. Biotinylated goat anti-mouse immunoglobulin (Ig)G was used as a linker. After washing, peroxidase-conjugated streptavidin was applied, and the sections were stained with diaminobenzidine. For the sections used to study p21, DAB staining was enhanced by incubation with methenamine silver solution for 5 min at 60°C (Peacock et al., 1991).
Western blotting
Villous and decidual tissues were lysed in a lysis buffer [50 mM Tris–HCl, pH 8.0, 0.25M NaCl, 0.5% NP-40, 1 mM phenylmethylsulphonyl fluoride (Sigma), 1 mg/ml aprotinin (Boehringer Mannheim), 1 mg/ml leupeptin (Boehringer Mannheim), 20 mg/ml TPCK (Boehringer Mannheim)]. The lysates were centrifuged at 13 000 g for 20 min at 4°C and the supernatants were stored at –80°C. Extracts equivalent to 30 µg of total protein were separated by SDS–polyacrylamide gel electrophoresis (10% acrylamide) and transferred onto nitrocellulose membranes (Hybond TM-C super, Amersham, Bucks, UK). The membranes were blocked in TBST (0.2 M NaCl, 10 mM Tris, pH 7.4, 0.2% Tween-20) containing 5% non-fat dry milk and 0.02% NaN3 for 1 h, then incubated with mouse monoclonal antibodies against Bcl-2 (Dakopatts), p21 (Pharmingen), or ß-actin (Biomakor, Rehovot, Israel) in TBST containing 5% non-fat dry milk. The membranes were then incubated with sheep anti-mouse IgG (Amersham) in TBST containing 2% non-fat dry milk. Bound antibody was detected by means of an enhanced chemiluminescence (ECL) system (Amersham).
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In the TUNEL method, apoptotic nuclei showing DNA fragmentation are dark brown in colour. They were identified sparsely in the glandular cells of the colon polyp (positive control). All the nuclei were labelled in positive control sections that had been pretreated with DNase, but no staining was observed in the negative control sections. In villous tissue, a cluster of labelled nuclei was localized just outside the cytotrophoblast layer (Figure 1A,B
). In a haematoxylin and eosin stained mirror section of that used for the TUNEL method, the labelled nuclei were seen to be clustered, small, condensed, and bland, and they often exhibited a ghost-like, blurred appearance. Apoptotic nuclei were almost always demonstrated within perivillous fibrin-type fibrinoid; this intervened within the syncytiotrophoblast layer and was characterized by the presence of dense homogeneous eosinophilic fibrin-like material in the cytoplasm (Figure 1A,B). Apoptotic nuclei were not observed in intervillous matrix-type fibrinoids; these are cell islands located between the chorionic villi and encasing extravillous trophoblast cells. Apoptotic nuclei were seldom observed in the other subtypes of the trophoblast. These observations were made in all the villous and decidual tissues examined. In hysterectomy specimens, nuclear DNA fragmentation was often recognized sparsely in myometrial cells and vascular endothelial cells, but not in the intermediate trophoblast. Although positive findings indicating DNA fragmentation were also observed in necrotic areas, the staining was not confined to the nuclei, and it was observed rather diffusely, so such areas were distinguishable from the apoptotic nuclei present in fibrin-type fibrinoid.
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By immunohistochemistry, Bcl-2 expression was diffusely demonstrated in the cytoplasm of syncytiotrophoblast, but perivillous fibrin-type fibrinoid, which contained apoptotic nuclei, was negative for Bcl-2 (Figure 2
). The other subtypes of the trophoblast, including the trophoblast of the cell columns, cytotrophoblast, and intermediate trophoblast, were negative for Bcl-2. Lymphocytes in the decidual tissues were often positive for Bcl-2. The necrotic areas showing positive staining for DNA fragmentation were negative for Bcl-2. The syncytiotrophoblast was partly positive for p21 in the nuclei (Figure 3), but the fibrin-type fibrinoid was negative for p21. The cytotrophoblast was only focally positive for p21. Cells positive for p21 did not show the characteristic nuclear features shown by apoptotic cells, and the p21-positive cells were topographically not coincident with the apoptotic cells (Figure 3). Western blotting analysis demonstrated specific bands for Bcl-2 in villous tissue (Figure 4). Specific bands for p21 were weakly observed in villous tissue, but not in decidual tissue (Figure 4).
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Discussion |
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This study demonstrated that fibrin-type fibrinoid, which is located in the perivillous portion of the chorionic villi and is positive for fibrin (Frank et al., 1994
; Benirschke and Kaufman, 1995), contained a cluster of apoptotic nuclei showing DNA fragmentation, but was devoid of Bcl-2 expression, and that the syncytiotrophoblast, which did show diffuse Bcl-2 expression, did not show apoptotic changes. In other words, there was an inverse relationship between apoptosis and Bcl-2 expression in both fibrin-type fibrinoid and syncytiotrophoblast. Nelson (1996) demonstrated by electron microscopy that nuclei within fibrin-type fibrinoid possessed features characteristic of apoptosis. On the other hand, Kokawa et al. (1998a,b) reported that in the first trimester normal placenta, apoptotic nuclei were localized predominantly to the cytotrophoblast lineage. In the present study using the TUNEL method, we found that when nuclear DNA fragmentation was detected, it was almost always in fibrin-type fibrinoid, and fibrin-type fibrinoid almost always showed nuclear DNA fragmentation. Moreover, syncytiotrophoblastic nuclei in the fibrin-type fibrinoid labelled by the TUNEL method generally showed chromatin condensation, a characteristic early sign of apoptosis (Kerr et al., 1994). To judge from these results, nuclear DNA fragmentation seems to occur specifically in fibrin-type fibrinoid.
Apoptosis characteristically affects scattered single cells, whereas necrosis affects groups of adjoining cells (Kerr et al., 1994). It is generally considered that clustered nuclei showing DNA fragmentation, as demonstrated here in fibrin-type fibrinoid, correspond to necrosis rather than to apoptosis (Kerr et al., 1994). In this study, however, the number of nuclei showing DNA fragmentation within fibrin-type fibrinoid was much smaller than that observed in areas of necrosis, and the histological features of the nuclei were compatible with those of typical apoptotic cells (Kerr et al., 1994; Cidlowski et al., 1996), not with those of necrotic cells. Further, the fibrin-type fibrinoid was never associated with an inflammatory reaction such as is usually seen in the process of necrosis (Kerr et al., 1994). Moreover, in the materials studied here, the positive staining in necrosis was dispersed and not confined to the nuclei, unlike that in the fibrin-type fibrinoid, so that apoptosis and necrosis were easily discriminated. Consequently, we believe that the nuclear DNA fragmentation seen here in the fibrin-type fibrinoid resulted from apoptosis. Since the fibrin-type fibrinoid is located adjacent to syncytiotrophoblast and, like syncytiotrophoblast, contains multiple nuclei, we suppose that the fibrin-type fibrinoid is derived from syncytiotrophoblast, a supposition also made in previous reports (Frank et al., 1994; Nelson, 1996).
Bcl-2 is a strong inhibitor of apoptosis and is thus generally expressed in highly proliferating cells in which apoptosis needs to be avoided. As shown in this study, as well as in previous reports (Sakuragi et al., 1994; Lea et al., 1997; Marzioni et al., 1998; Quenby et al., 1998), Bcl-2 is diffusely expressed in syncytiotrophoblast. In particular, Marzioni et al. (1998) reported discontinuities in the intensity of Bcl-2 expression in perivillous fibrinoid, a finding compatible with the results of the present study. However, the proliferative activity of syncytiotrophoblast is extremely low, judging from the reported Ki-67 labelling index (Ichikawa et al., 1998). Thus, we might need to seek a different biological function for the Bcl-2 expression seen in syncytiotrophoblast. The syncytiotrophoblast is a peculiar multinuclear cell that is believed to be formed following the fusion of cytotrophoblast cells (Mazur and Kurman, 1994; Benirschke and Kaufman, 1995). One of the most significant morphological characteristics of syncytiotrophoblast is that it possesses multiple nuclei sharing the same cytoplasm. As we mentioned above, apoptosis usually occurs in scattered single cells in the case of typical mononuclear cells. In fact, in the present study, apoptotic nuclei were usually observed in a cluster within the fibrin-type fibrinoid. Therefore, it can be speculated that the nuclear multiplicity shown by syncytiotrophoblast might in some way be related to the diffuse Bcl-2 expression seen in syncytiotrophoblast. On the other hand, no evident Bcl-2 expression was observed in the fibrin-type fibrinoid containing clustered apoptotic nuclei. If the syncytiotrophoblast, as a multinuclear giant cell in which the nuclei all share the same cytoplasm, tends to undergo spreading nuclear DNA fragmentation, such apoptotic changes would need to be controlled by certain suppressors. Although apoptosis is regulated by a number of substances (Hale et al., 1996), diffuse Bcl-2 expression might play a major part in avoiding possible excessive nuclear degradation in syncytiotrophoblast by virtue of its apoptosis-inhibiting action. We therefore speculate that the major role of Bcl-2 in syncytiotrophoblast may be to inhibit the spread of apoptosis to the other nuclei sharing the same cytoplasm, rather than to immortalize the syncytiotrophoblast. Lea et al. (1997) reported that Bcl-2 was expressed in large granular CD56-positive lymphocytes in decidua, so the weak bands detected for decidual tissue by Western blot analysis could be due to these Bcl-2-positive lymphocytes.
Cell growth arrest induced by p21 is another negative regulatory mechanism that controls cell proliferation (El-Deiry et al., 1993; Harper et al., 1993). p21 is an inhibitor of G1 CDKs, and its overexpression eventually inhibits DNA synthesis and arrests cell proliferation (El-Deiry et al., 1993; Harper et al., 1993). Quenby et al. (1998) recently reported an intense p21 expression in the cytoplasm of the villous cytotrophoblast, a result inconsistent with ours (infrequent expression in the nuclei of the syncytiotrophoblast). Our experience with the human endometrium is that p21 expression is localized in the nuclei of the glandular cells, not in the cytoplasm (Toki et al., 1998). The detection of weak bands for p21 in villous tissue by Western blot analysis also supports the staining results obtained in the present study. p21 expression was partly identified in syncytiotrophoblast, but the localization of the p21-positive cells was not coincident with that of the apoptotic cells. We recently reported that there was also a discrepancy between the localization of apoptotic cells and that of p21-positive cells in the human endometrium (Toki et al., 1998). Not only apoptosis, but also p21 is induced by p53 (El-Deiry et al., 1993; Hale et al., 1996). In the present study, the localization of p53 was not investigated because wild-type p53 can barely be detected by immunohistochemistry (Vojtesek et al., 1992; Yin et al., 1993). Partial p53 expression has been demonstrated in the cytotrophoblast and in the trophoblast of the cell columns (Haidacher et al., 1995; Ichikawa et al., 1998; Quenby et al., 1998), although it is not clear whether the expressed p53 was wild-type or mutated. In either case, the immunolocalization of p53 was not coincident with the localization of the apoptotic cells or of the p21-positive cells, suggesting that p53 overexpression may not be related directly to apoptosis or cell cycle arrest in the human trophoblast.
Activation of p53 leads to either the cessation of DNA replication (growth arrest) for long enough for the genomic DNA to be repaired, or to the death of the cell (apoptosis), which prevents the damage-induced mutation being incorporated into the genome (Lu et al., 1998). This being so, it can be speculated that if p53 becomes activated in syncytiotrophoblast, the diffuse Bcl-2 expression might inhibit the induction of apoptosis by the activated p53. Taken together, the available evidence seems to suggest that there may be two regulatory mechanisms controlling apoptosis and cell cycle arrest in syncytiotrophoblast: suppression of apoptosis by diffuse Bcl-2 expression and growth arrest by p21. However, at present this idea is speculative, and since the precise biological role of the apoptosis occurring in fibrin-type fibrinoid is as yet unknown, further investigation of this subject would seem to be necessary.
In conclusion, in the human trophoblast, apoptosis occurs specifically in fibrin-type fibrinoid, which is devoid of Bcl-2 expression, whereas the syncytiotrophoblast, which shows a diffuse expression of Bcl-2, does not show apoptotic changes. p21 expression is observed to some extent in the syncytiotrophoblast, but not in fibrin-type fibrinoid. These results suggest that Bcl-2 may play an important role in preventing apoptosis in the syncytiotrophoblast, which may be necessary to prevent any DNA degradation that occurs from being spread to other nuclei within this multinuclear cell. p21 may also play a role as an inhibitor of cell proliferation in the syncytiotrophoblast.
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3 To whom correspondence should be addressed
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Submitted on July 17, 1998; accepted on December 18, 1998.
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