- Open Access
Expression of Bcl-2 and p53 at the fetal-maternal interface of rhesus monkey
© Wei et al; licensee BioMed Central Ltd. 2005
- Received: 17 September 2004
- Accepted: 14 January 2005
- Published: 14 January 2005
To study the apoptosis and its mechanism at the fetal-maternal interface of early gestation, localization of apoptotic cells in the implantation sites of the rhesus monkey on day 17, 19, 28 and 34 of pregnancy were first examine by using the TUNEL technique. The expression of Ki67, a molecular marker of proliferating cells, and two apoptotic proteins, B cell lymphoma/leukaemia-2 (Bcl-2) and P53, were then studied by immunohistochemistry. Apoptotic nuclei were observed mainly in the syncytiotrophoblast. Ki67 was confined almost exclusively to cytotrophoblasts. The localization of Bcl-2 protein follows that of the apoptotic nuclei and its expression level increased as the development of the placenta progressed on. P53 was detected to some extent in cytotrophoblasts and syncytiotrophoblast covering the basal feet of the anchoring villi during the late stage of placentation. Based on these observations, it might be suggested that Bcl-2 could be possible to play an interesting role in limiting degree of nuclear degradation and sustaining cell suvival in the multi-nucleated syncytiotrophoblast cells during early pregnancy, and P53 could also be essential in regulating the trophoblastic homeostasis by controlling its proliferation or apoptosis.
- Cell Column
- Terminal Deoxynucleotidyl Transferase
- Implantation Site
- Apoptotic Nucleus
- Placental Villus
Apoptosis plays important roles in placentation and embryonic development . The cells undergoing apoptosis have characteristic structural changes in the nucleus and cytoplasm. The nuclear disintegration involves DNA cleavage into oligonucleosomal length DNA fragments [2–4], and the DNA fragments can be detected by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end-labelling (TUNEL) technique. Expressions of apoptotic regulatory molecules, such as Fas, Fas ligand, P53, and the proteins of Bcl-2 family, have been reported in human placenta [5–8]. Bcl-2 and P53 are two of the key players in the apoptotic signaling cascades. Bcl-2, a proto-oncogene first discovered in human follicular lymphoma , is involved in the inhibition of apoptosis and the survival of a variety of cell types . Bcl-2 protein is located in the membranes of endoplasmic reticulum, nuclear envelope, and mitochondria. Over-expression of Bcl-2 suppresses apoptosis by preventing the activation of caspases that carry out the process. P53 is well known as a tumor suppressor. It is a transcription factor that induces apoptosis mainly through inducing the expression of a batch of redox-related genes  and the down-regulating Bcl-2 .
The expression of Bcl-2 and P53 human placenta has been studied [1, 13]. However, their cellular distribution in the implantation site at early stage of pregnancy has not been reported. Because the monkey and the human share a very similar implantation process in terms of timing, morphological changes, and cell types involved , we aimed, in the present study, to investigate the expression, localization of Bcl-2 and P53 in the implantation site of the rhesus monkey, in order to gain some insights to the mechanism of time-dependent apoptosis occurring at the fetal-maternal interface.
Healthy adult male and female rhesus monkeys (Macaca mulatta) were purchased from the monkey colony of the Primate Research Center (PRC), Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS). All experimental procedures were approved by the Animal Ethics Committees of both the Institute of Zoology and PRC. The animals were caged individually and were evaluated daily by visual examination of the perineum for menses, with the onset of menses defined as Day 1 of the menstrual cycle. Adult female monkeys with regular menstrual cycles of approximately 28 days were chosen for this study. Female monkeys on Day 11 of their menstrual cycle were caged with a male monkey of proven fertility from previous mating for 3 days. Vaginal smears were examined the next morning for the presence of sperm. The day when the smear was detected as positive for sperms was designated as Day 1 of pregnancy (D1). The presence of a conceptus was confirmed by ultrasound examination. The monkeys were anesthetized by pentobarbital sodium (3 animals each group), and the uteri were removed surgically from early villous to villous placenta stages: on D17, D19, D28 and D34 of pregnancy respectively and cut into pieces, the specimens were quickly washed in cold phosphate-buffered saline (PBS) to remove adherent blood, then placed in cold 4% paraformaldehyde fixative for 16 h at 4°C and further processed through graded dehydration, clearing and embedding in paraffin for immunohistochemistry and TUNEL assay. Part of the specimen was cryopreserved at -70°C for Western blot analysis.
Western blot was done as previously described  with slight modifications to verify the cross-reactive specificity of the antibodies with the monkey tissue. The tissue of implantation sites on D34 of pregnancy was homogenized and the supernatant (50 μg) from centrifugation was run on a 10% SDS-PAGE gel under reduced conditions. After being transferred to the polyvinylidene difluoride membrane, individual lanes were cut and blocked with 5% nonfat milk/PBS for 1 h, followed by incubation at 20°C for 1 h with the primary antibodies (IgG, 0.2 μg/ml) in 5% milk/PBS. The membranes were washed three times, 5 min for each, in 5% milk/PBS and incubated with HRP-conjugated horse anti-mouse IgG (0.2 μg/ml, for Bcl-2) or HRP-conjugated goat anti-rabbit IgG (0.04 μg/ml, for P53) in 5% milk/PBS for 1 h respectively. The membranes were washed in PBS three times 5 min for each, followed by 10 min of incubation with SuperSignal® West Pico substrate, then exposed on x-ray film. For negative controls, primary antibodies were replaced with normal IgG of the same concentration and origin.
Apoptotic cells were identified by using the TUNEL technique [1, 16]. The procedure was slightly modified based on Gao et al.  as the following. Deparaffinized and hydrated 4 μm sections were first treated with 10 μg/ml proteinase K at 37°C for 20 min, and then subjected to 3'-end-labelling of the DNA with 1 μM DIG-11-dUTP and 1 U/μl TdT at 37°C for 1 h. The sections were washes three times in Tris buffer, and incubated with blocking buffer (100 mM Tris, 150 mM NaCl, pH 7.5, and 1% blocking reagent) for 30 min at room temperature. Next, sections were incubated with the primary AP-conjugated anti-DIG antibody (1:500 in 1% blocking reagent, 100 mM Tris, and 150 mM NaCl, pH 7.5) at room temperature for 2 h, and then washed with Tris buffer. Staining was developed using the standard substrates NBT (337.5 μg/ml) and BCIP (175 μg/ml). Negative controls were similarly processed with the omission of TdT.
Serial 4 μm sections of tissue were deparaffinized and rehydrated through degraded ethanol. Antigen retrieval was performed by incubating the sections in 0.01 M citrate buffer (pH 6.0) at 98°C for 20 min followed by cooling at room temperature for 20 min. Non-specific binding was blocked with 5% (v/v) normal goat serum in PBS for 1 h. The sections were incubated with primary antibodies specific for P53 (1 μg/ml), Bcl-2 (2 μg/ml) or Ki67 (2 μg/ml) respectively in 2% goat serum overnight at 4°C. Sections were then washed three times with PBS (10 min each) and incubated with biotinylated secondary antibody (2 μg/ml) at RT for 30 min. 3 × 10 min successive washes were followed by incubation with avidin-AP complex (1:200, RT, 20 min). Sections were developed with standard substrates (337.5 μg/ml NBT and 175 μg/ml BCIP) or Vector Red AP substrates according to the manufacturer's protocol after another three washes. Endogenous AP activity was inhibited by supplement of 1 mM levamisole into substrate. The sections stained with Vector Red substrates were counter-stained using haematoxylin. Sections incubated with normal IgG instead of primary antibody served as negative controls.
A double immunostaining technique using the antibodies to cytokeratin and actin was performed to localize the extravillous endovascular trophoblast cells. De-paraffinized sections were incubated with 3% H2O2 in methanol for 10 min at room temperature to quench endogenous peroxidase after antigen retrieval treatment as described above. To detect the cytokeratin signal, the sections were washed (3 × 10 min in PBS), blocked for nonspecific signals, incubated sequentially with primary anti-human cytokeratin anbibody (1 μg/ml, RT, 1 h), secondary biotinylated goat anti-rabbit IgG (2 μg/ml, RT, 30 min), and avidin-peroxidase complex (1:200, RT, 20 min), and developed with DAB substrate solution in a similar way as described above. To detect actin signal, the procedure was repeated one more time with anti-human actin antibody (1 μg/ml, RT, 2 h) as primary antibody, AP conjugated horse anti-mouse IgG (1 μg/ml, RT, 40 min) as secondary antibody, and Vector Red developing AP substrate. As a result, the trophoblast cells were labeled brown and the blood vessel wall red.
Placental samples from three individual monkeys for each group were analyzed. Experiments were repeated at least three times, and one representative from at least three similar results was presented. The mounted sections were examined using a Nikon microscope. For Ki67, the percentages of immunoreactive cells were assessed on at least 2000 cells in each tissue section; For TUNEL, the percentages of positive nuclei were assessed out of at least 2000 nuclei in each tissue section; For assessment of Bcl-2 staining intensity in cells of different compartments, semi-quantitative subjective scoring was performed by three blinded investigators using a 4-scale system with "-"= nil; "+/-"= weak; "+" = moderate; and "++" = strong as described by Yue et al. .
Apoptosis in implantation site of early pregnancy
Proliferative activity in implantation site at early pregnancy
Bcl-2 expression in implantation site at early pregnancy
Semi-quantitative assessment of the immunohistochemical staining of Bcl-2 in the placenta of rhesus monkey.
Syncytiotrophoblast lining the villi
Syncytiotrophoblast covering the cell column
cytotrophoblast lining the villi
P53 expression in implantation site at early pregnancy
For the first time in present study, we investigated the expression of Bcl-2 and P53 in relation to apoptosis at the fetal-maternal interface of rhesus monkey at the very early stages (D17-D34) of gestation. Villous trophoblasts consist of cytotrophoblasts and syncytiotrophoblast. While cytotrophoblasts possess a brisk mitotic activity during the first trimester of gestation in human, the syncytiotrophoblast is incapable of cell division despite of a metabolic activity . This fact implies that cell proliferation is differently regulated in these two cell types. The reports on the type of trophoblast cells undergoing apoptosis in the first trimester are controversial [1, 21, 22]. Our results further cleared that the apoptotic nuclei were distributed mainly in the syncytiotrophoblast at the early stages and in the cytotrophoblasts within the cell columns at later stages of pregnancy.
P53 was partly identified in some nuclei of the syncytiotrophoblast with the same position of apoptotic nuclei, in the basal feet of the anchoring villi in particular, but it is not clear whether the P53 was co-localized with the apoptotic signals. Activation of P53 in some cell types leads to either the cessation of cell growth or apoptosis . Therefore, P53 protein might be related to cell cycle arrest or apoptosis in syncytiotrophoblast during early stage of placentation. Low level of P53 staining was detected in the cytotrophoblasts during the earlier stages of gestation (D17 and D19). However, at the later stages (D28 and D34), the expression was observed predominantly in the nuclei of cytotrophoblasts. The presence of P53 in cytotrophoblast in the primate was consistent with that observed in the human first trimester placenta . Indeed, the TUNEL staining showed that the apoptosis seldom happened in the cytotrophoblast, with the exception of cytotrophoblast at proximal tip of cell columns during later stages of placentation (D28, D34) where a high proliferative activity and P53 expression were detected. This finding supports the hypothesis that a physiological upregulation of the P53 tumour suppressor gene might be a mechanism for controlling excessive trophoblastic proliferation in normal placentation [26, 28].
It is known that early pregnancy is unique in its methods of cell proliferation control, the existing data suggest that some growth factors and transcription factors from the embryo and endometrium, such as CSF-1, VEGF, and transcription factors of the helix-loop-helix family, provide at least part of this control . In addition, other studies found maternal age and some diseases, such as diabetes can also influence the apoptotic and proliferative activities in trophoblast cells [30, 31]. Further investigations are required to uncover which endocrine event regulates the expression of Bcl-2 and P53.
This study was supported by WHO/Rockefeller Fundation Project (RF96020#78), the National Science Fundation of China (30270196), Chuang-Xin program of Chinese Academy of Sciences (KSCX-2-SW-201/IOZ-7), and the Chinese "973" Program (G1999055901).
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