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a Research Laboratory on Reproduction and
b Laboratory of Pharmacology, Université Libre de Bruxelles (ULB), B-1070 Bruxelles, Belgium
c Unité Vétérinaire, Université Catholique de Louvain (UCL), B-1348 Louvain-La-Neuve, Belgium
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The incidence of apoptotic nuclei (< 1%) as evidenced by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method, and the histological distribution of immunoreactive Bcl-2, Bax, and Bcl-x proteins, were similar in placentae collected after delivery and before the onset of labor and in placental explants maintained overnight at 4°C in a minimal salt-Hepes medium. By contrast, 28% of nuclei contained fragmented DNA when placental explants were incubated overnight at 37°C. This marked increase was associated with a decrease in the intensity of the Bcl-2 immunostaining and an increase in the intensity of Bax and Bcl-x immunostaining.
In conclusion, the present study clearly evidences the presence of an active apoptotic machinery in term placental cells that is not involved in normal parturition.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The occurrence of apoptosis has been described in many human reproductive tissues including the uterine epithelium [5], the mammary gland [6, 7], the testicle [8, 9], the ovary [10, 11], and the placental villi [12]. The incidence of apoptosis in human placental tissue, as evidenced by studies on DNA fragmentation levels and immunolocalization of Bcl-2, was reported to progressively increase throughout pregnancy and until close to delivery [13, 14]. However, the question of activation of the apoptotic machinery in the initiation of labor remains to be elucidated. We hypothesized that the incidence of apoptosis might be increased in relation to a decrease in placental perfusion during uterine contractions. Under such physiological conditions, the balance between anti- and pro-apoptotic protein expression at the placental level might be altered in close relation to changes in DNA fragmentation levels. The purpose of the study was to investigate variations of apoptosis-related signs on histological sections of human term placentae obtained before the onset of labor or after delivery. Moreover, ex vivo incubations of placental explants were performed in a minimal salt-balanced medium in order to examine the reactivity of the placental apoptotic machinery in terms of DNA fragmentation levels and expressions of immunoreactive proteins from the Bcl-2 family.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Morphologically normal term placentae (37–41 wk of gestation) were collected after vaginal delivery (n = 5) and immediately transferred to the laboratory. For each placenta, large pieces of villous tissue free of visible infarct, calcification, or hematoma were sampled from at least 5 cotyledons, midway between the chorionic and basal plates. These inner parts of cotyledons were cut into multiple fragments (0.5 g) that were thoroughly rinsed in a cold physiological medium. Fragments (n 
5) were either fixed in a 10% formaldehyde-PBS solution for 48–96 h for further histological observations or stored at -20°C until homogenization. PBS buffer contained 40 mM Na2HPO4, 10 mM KH2PO4, and 120 mM NaCl with a resulting pH of 7.2. All reagents were of analytical grade.
The remaining fragments were incubated overnight (17–24 h) at 4°C (n 5) or 37°C (n 5) in flasks containing 200 ml of incubation medium continuously gassed under a humidified atmosphere of 100% O2. The incubation medium consisted of a Hepes-buffered physiological salt solution (pH 7.4) composed of 139 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM Hepes, and 4.2 mM glucose. The medium was supplemented with 0.5% (w:v) BSA (fraction V; Boehringer Mannheim, Mannheim, Germany), 50 IU/ml penicillin, and 50 µg/ml streptomycin (GIBCO BRL, Gaithersburg, MD). After overnight incubation, placental fragments were rinsed and then either fixed in the 10% formaldehyde-PBS solution or stored at -20°C until homogenization.
For histological determinations, 5-µm paraffin sections from placentae obtained both before and after the onset of labor were placed onto silane-coated slides and rehydrated.
DNA fragmentation was detected on tissue sections by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) method [15, 16]. This method was applied with special attention to tissue processing (short-time fixation) and to pretreatment (heating) in order to minimize loss in sensitivity, as previously assessed [17, 18]. Briefly, sections were pretreated by heating for 1 h at 60°C in distilled water, then bathed for 15 min in 50 mM aqueous solution of NH4Cl at 20°C. Endogenous peroxidase was inactivated by covering the sections for 10 min with 0.6% (v:v) H2O2 containing 0.1% (w:v) NaN3. The slides were immersed for 15 min at 37°C in a TdT buffer (pH 7.2) composed of 25 mM Trizma base, 150 mM sodium cacodylate, 1 mM cobalt chloride, and 0.25 mg/ml BSA. The sections were then incubated for 120 min at 37°C in the TdT buffer supplemented with 0.37 U/ml TdT, 2.5 pmol/ml biotin-1,6-dUTP, and 1 nmol/ml dATP (Boehringer Mannheim). The reaction was stopped by rinsing of slides three times in PBS. Finally, the sections were covered with a blocking PBS buffer containing 5% (v:v) newborn calf serum (Sigma Chemical Co., St. Louis, MO) and incubated for 20 min with biotinylated streptavidin-peroxidase complex (ABComplex/HRP Dako kit; Dako, Glostrup, Denmark). For each set of experiments, a positive control (testicular tissue from hamster, provided by Dr. Nonclercq, Department of Histology, Université de Mons-Hainaut, Mons, Belgium) was systematically included. In the absence of the TdT enzyme, positive nuclei were never observed within testicular and placental tissue.
Immunohistochemical detection of proteins from the Bcl-2 family was performed using the peroxidase-antiperoxidase technique [19]. Briefly, hydrated slides were bathed for 10 min in methanol and then for 10 min in 0.3% H2O2 to quench endogenous peroxidase activity. Sections were incubated with 2% (v:v) normal sheep serum in PBS to block nonspecific immunoreactive sites. The incubation step was carried out at 4°C overnight with the primary antibody diluted in PBS buffer supplemented with 1% (v:v) normal sheep serum, 1% (v:v) normal human serum, and 1% (w:v) BSA. According to data provided by the manufacturers, the monoclonal mouse anti-human Bcl-2 antibody (Boehringer Mannheim) binds specifically to the bcl-2 oncogene product. The polyclonal rabbit anti-human Bax antiserum (Pharmingen, San Diego, CA) does not cross-react with the closely related Bcl-2 protein. The polyclonal rabbit anti-human Bcl-x antiserum (Pharmingen) recognizes a region common to both Bcl-xLong and Bcl-xShort proteins. The peptide used as immunogen corresponds to a unique domain that lacks homology with other known members of the Bcl-2 protein family. Working serial dilutions ranged from 1:20 to 1:80 for the anti-Bcl-2 antibody and from 1:100 to 1:400 for the anti-Bax and anti-Bcl-x antisera. Sections were incubated for 10 min at room temperature with peroxidase-labeled secondary antibodies (sheep anti-mouse or -rabbit IgG; Dako) diluted 1:100 in the PBS buffer containing sera. No immunostaining could be observed when sections were incubated with the PBS buffer instead of the primary antibody.
Peroxidase activity was evidenced by DAB (3,3'-diaminobenzidine; Fluka Chemica, Buchs, Switzerland) staining [19]. Slides were counterstained with methyl green or toluidine blue, dehydrated, and then mounted with a coverslip for histological examination. All tissue sections from the same placenta were processed in the same experiment for each apoptosis-related marker. The intensity and incidence of the peroxidase activity staining were microscopically evaluated by two independent observers using a micrometer reticle (Omnilabo, Brussels, Belgium). For each placenta, the observations were processed on five fragments (one per slide), and 10 fields (at x200 magnification) per slide were taken into account. For each slide, the number of TUNEL-positive nuclei was expressed as a percentage of the total nuclei counted (approximately 10 000). In addition, the number of villous sections containing either TUNEL-positive nuclei or Bcl-2 immunostaining was expressed as a percentage of the total villi observed (approximately 400 per fragment). Only villous sections ranging from 125–625 µm2 were considered for this assessment. Statistical differences between conditions were analyzed using ANOVA followed by a Scheffe multiple range test.
In order to further assess variations in the intensity of immunostaining as observed using immunohistochemistry, dot-blot analyses were performed using serial dilutions of placental homogenates prepared from fragments incubated at 4°C or 37°C. Briefly, explants were sonicated after a 40-min immersion in a cold lysis buffer containing 50 mM Hepes, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 2.5 mM PMSF, and 1 µg/ml aprotinin. Homogenates were centrifuged for 10 min at 4000 x g. Protein contents in supernatants were determined using Lowry's protein assay [20]. All supernatants were adjusted in the lysis buffer to 1.5 mg/ml total protein; they were then diluted in SDS sample buffer made of 0.125 M Tris-HCl (pH 6.8) enriched with 5% SDS and 20% sucrose [21]. Successive dilutions (375–11.7 µg/ml sample buffer) were directly adsorbed onto nitrocellulose sheets (1 µl/dot). Nitrocellulose was washed twice in PBS and blocked in 4% ovalbumin in PBS for 1 h, then reacted for 48 h in a humid chamber with primary antibodies as used in immunocytochemical procedures (mouse anti-human Bcl-2, 1:40; rabbit anti-human Bax, 1:200; rabbit anti-human Bcl-x, 1:200). Nitrocellulose sheets were incubated with an alkaline phosphatase-conjugated secondary antibody (sheep anti-mouse IgG, 1:350; sheep anti-rabbit IgG, 1:750; BioSys, Compiègne, France) in PBS with 0.05% Tween 20 for 60 min. Alkaline phosphatase activity was evidenced by incubating nitrocellulose with a Tris solution containing nitro blue tetrazolium (325 µg/ml), 5-bromo-4-chloro-3-indolylphosphate (136 µg/ml), and phenazine methosulfate (13.4 µg/ml). No immunostaining was observed when the primary antibody was omitted. The intensity of the phosphatase activity was semiquantitatively assessed using an arbitrary comparative scale graded from "-" for no staining to "+++" for the most intensely stained dots. The concordance between results obtained by two independent observers was very good, with an interobserver kappa value of 0.88 [22].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In hematoxylin/eosin-stained sections from our placental preparations, the large majority of nuclei were considered morphologically normal, but some, randomly distributed in the section, presented evidence of shrinkage and condensation of chromatin. No necrotic areas were detected.
Bcl-2 immunostaining in term placentae was observed exclusively in the cytoplasm of the syncytiotrophoblastic layer (Fig. 1B, left). Nuclei were never immunostained for Bcl-2. No Bcl-2 staining was detected in cells from the villous stroma and fetal capillaries. A positive trophoblastic layer could be observed in 85% of all observed villi (Table 1). The presence of this immunostaining was not related to the area of the villous section.
The immunostaining obtained with the anti-Bax serum was distributed in the cytoplasm of the syncytiotrophoblastic layer as well as of cells from the stroma and from fetal capillaries (Fig. 1C, left). This cytoplasmic distribution of Bax staining was observed in all villi. Nuclei were never immunostained for Bax.
The cellular distribution of the Bcl-x immunostaining was similar to that obtained with the Bax antiserum. However, in addition to this cytoplasmic immunostaining, some nuclei were also stained (Fig. 1D, left). As observed for the Bax immunoreactivity, Bcl-x staining was detected in all villi.
Within placentae obtained after elective cesarean sections, the distribution of nuclei evidenced by the TUNEL method, as well as the distribution and the intensity of the Bcl-2, Bax, and Bcl-x immunostaining, was similar to findings for placentae collected after normal delivery. The incidence of stained nuclei and the percentage of villi containing trophoblastic Bcl-2 immunostaining were not significantly different from those observed in placentae obtained after vaginal deliveries (Table 1).
In placental explants incubated overnight at 4°C, the cellular distribution of TUNEL-positive nuclei, as well as the intensity and distribution of immunoreactive Bcl-2, Bax, and Bcl-x, was again not different from observations in freshly delivered placentae.
The proportion of stained nuclei, as well as the percentage of villi with Bcl-2-positive trophoblasts, was statistically similar to values estimated in the same placentae fixed immediately after delivery (Table 1).
When placental explants were maintained overnight at 37°C, a completely different picture was observed (Fig. 1, right). Indeed, the incidence of nuclei detected by the TUNEL method in all villous cell types (trophoblast, stroma, vascular endothelium) (Fig. 1A, right) reached 28% of all nuclei (Table 1). Among fragments from the same placenta, the ratio between the maximum and minimum incidences of stained nuclei could reach 3. Villi containing stained nuclei represented 74% of all villi (Table 1). A large number (36%) contained more than 50% of positive nuclei in the trophoblastic layer. The remaining villi, without any stained nuclei (26%), were randomly distributed in the section.
The intensity of Bcl-2 immunostaining, exclusively distributed in the cytoplasm of the syncytiotrophoblastic layer (Fig. 1B, right), was clearly reduced when compared to that observed in the same placenta fixed immediately or after an overnight incubation at 4°C. In addition, the percentage of observed villi presenting trophoblastic Bcl-2 staining was decreased (Table 1).
At identical dilutions of the Bax antiserum, the intensity of immunostaining (Fig. 1C, right) was more marked in placental explants maintained overnight at 37°C than in those from the same placenta fixed after delivery or maintained at 4°C. With the highest dilution of antiserum (1:400), Bax staining became weaker in the cytoplasm but remained intense at the perinuclear area of numerous syncytiotrophoblastic and stromal cells (data not shown).
Using the Bcl-x antiserum, the cytoplasmic distribution in the syncytiotrophoblastic, stromal, and endothelial cells (Fig. 1D, right) was also more intense after an incubation at 37°C than in placental pieces fixed directly after delivery or incubated at 4°C. Some immunostained nuclei within the syncytiotrophoblast and the villous stroma were also observed in fragments incubated at 37°C.
In order to semiquantify variations in the intensity of immunostaining observed in immunohistochemistry, homogenates, prepared from placental samples incubated at either 4° or 37°C, were adsorbed as 1-µl dots onto nitrocellulose. The immunoreactive signals for Bcl-2, Bax, and Bcl-x corresponding to samples homogenized after an incubation at 4°C were clearly different from those after a 37°C incubation (Table 2). At 37°C, the immunoreactivity for Bcl-2 was less intense; using Bax and Bcl-x antisera, the signals were more intense.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our study also reveals that apoptosis is markedly induced when placental tissue is maintained at 37°C in a minimal salt-Hepes medium. Indeed, the increased incidence of fragmented DNA was accompanied by modifications in the immunoreactivity for proteins from the Bcl-2 family, as observed using immunohistochemical and dot-blot approaches. Immunostaining for the anti-apoptotic Bcl-2 protein decreased after incubation of placental explants at 37°C. Concomitantly, the immunoreactivity for the pro-apoptotic Bax protein and the Bcl-x proteins was increased in all cells of the villi and in homogenates from explants incubated at 37°C. This suggests a modification of the balance between suppressors and inducers of apoptosis within the Bcl-2 family [4]. The functions of proteins from the Bcl-2 family have been reported to be modulated according to their ability to form homo- or heterodimer [26] and to their subcellular localization [27, 28]. A shift of the Bcl-x and Bax proteins from soluble cytosolic forms to forms associated with the perinuclear envelope has been reported in murine thymocytes involved in the apoptotic process [29]. Such a cytosol-to-membrane redistribution is compatible with our observations that immunoreactive Bcl-x and, to a lesser extent, immunoreactive Bax were concentrated in nuclear areas within trophoblastic and stromal cells from explants incubated at 37°C.
This experimentally induced apoptosis, detected in placental explants, was prevented by lowering temperature. Such a feature indirectly supports the view that in placenta as in other tissues, apoptosis is a metabolism-dependent process.
Incidentally, the overwhelming modifications of apoptosis-related markers (levels of DNA fragmentation and immunoreactive proteins from the Bcl-2 family) were not observed when placental explants were incubated overnight at 37°C in a "rich" culture medium such as RPMI 1640 (data not shown). This suggests the importance of environmental conditions on the intracellular regulation of apoptosis of the incubated tissue. Thus, our experimental model of explant incubations might be used to investigate effects of environmental conditions on the viability of the placental tissue.
Taken as a whole, these observations show that mechanisms of regulation of apoptosis are present in the placenta and are able to react concomitantly to an apoptosis-inducing environment. However, the viability of the placental tissue was not impaired during intermittent decreases in placental perfusion occurring in labor until delivery. Our findings support the importance of a balanced apoptosis regulatory process during normal pregnancy and, by extension, in placental physiopathology. It is tempting to speculate that some maternal or fetoplacental factors could be involved in the maintenance of cellular homeostasis until delivery. For instance, several growth factors such as epidermal growth factor, insulin growth factor, transforming growth factor alpha, and vascular endothelial growth factor are associated with the inhibition of apoptosis in various cellular systems [30–33].
In conclusion, the present study clearly evidences the presence of an active apoptotic machinery in term placental cells that is not involved in normal parturition.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Nadia Cirelli, Research Laboratory on Reproduction, CPi 626, Faculty of Medecine, Université Libre de Bruxelles, 808, Route de Lennik, B-1070 Bruxelles, Belgium. FAX: 32 2 555 63 56; ncirelli{at}ulb.ac.be
Accepted: March 15, 1999.
Received: June 10, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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