Introduction

Apoptosis plays important roles in placentation and embryonic development [1]. 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 [9], is involved in the inhibition of apoptosis and the survival of a variety of cell types [10]. 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 [Microscopic assessment

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. [18].

Results

Apoptosis in implantation site of early pregnancy

The TUNEL technique was used to identify cell types that underwent apoptosis in the implantation site of rhesus monkey on D17, D19, D28 and D34 of pregnancy. On D17 and D19, apoptotic nuclei were observed in the syncytiotrophoblast layer covering the basal feet of the anchoring villi (Figure 2 A, B, arrowhead) and in the villous stromal cells (Figure 2 A, arrow), but not in the cytotrophoblasts. The positive nuclei in the syncytiotrophoblast was only about 0.06%. On D28 and D34, the apoptotic nuclei were present in the syncytiotrophoblast covering the villi (Figure 2 C, D), in the villous stromal cells (Figure 2 C, D, arrow), in the syncytiotrophoblast layer covering the basal feet of the anchoring villi (Figure 2 E), and in the cytotrophoblasts within the cell columns (Figure 2 F). On D28, the percentage of TUNEL-positive nuclei in the syncytiotrophoblast was 0.21%. As pregnancy progresses, the percentage increased to 0.34% on D34. In maternal compartment, a lot of apoptotic nuclei were detected in the stromal cells (Figure 2 G) and glandular epithelium (Figure 2 H).

Figure 2
figure 2

Apoptosis detected by TUNEL at the implantation sites of the rhesus monkey on D17 (A), D19 (B), D28 (C, G) and D34 (D, E, F, H) of gestation. Apoptotic nuclei were stained dark. Arrowhead and arrow in panel A – D indicated the nuclei of syncytiotrophoblast and villous stromal cells respectively. The insets in C and D showed the positive nuclei under a higher magnification. Note the syncytiotrophoblast layer covering the basal feet of the anchoring villi in E and the cell columns in F. G and H represent the stromal cells and glandular epithelial cells respectively in the endometrium. I was the negative control. St, syncytiotrophoblast; CT, cytotrophoblast; Vi, placental villi. Scale bars represent 50 μm.

Full size image

Proliferative activity in implantation site at early pregnancy

Ki67 is a protein expressed in cycling cells from G1 to M phases and is widely used as a roliferative marker (19, 20). As shown in Figure 3, Ki67 was expressed in the cytotrophoblasts and the villous stromal cells, but not in the syncytiotrophoblasts. As pregnancy progresses, the percentage of Ki67-positive cytotrophoblast cells lining the villi decreased from more than 85% on D17 to less than 25% on D34 (Panel A-D, and E, F for a higher magnification). However, the cytotrophoblasts at the proximal tip of cell columns remained highly proliferative (more than 70%) at all stages (Panel G and H).

Figure 3
figure 3

The proliferating activity revealed by Ki-67 immunostaining at implantation sites of the rhesus monkey on D17 (A, E, G), D19 (B), D28 (C) and D34 (D, F, H) of gestation. Panels A-D were under a lower magnification. Ki-67 protein was stained red, and nuclei blue. E and F were the placental villi under a higher magnification. G and H were the anchoring villi under a higher magnification. Vi, placental villi. ST, syncytiotrophoblast. CT, cytotrophoblast. Sc, stromal cell. En, endometrium. Scale bars represent 100 μm.

Full size image

Bcl-2 expression in implantation site at early pregnancy

In order to study the mechanisms of the apoptosis observed at the fetal-maternal interface, the expression of Bcl-2 was investigated by using immunohistochemistry. At the early stages of placentation (D17, D19), Bcl-2 was only detected in the syncytiotrophoblast covering the cell columns (Figure 4, A and 4B) and the extravillous cytotrophoblast (Figure 4C, arrow). At the later stages (D28, D34) it was detected in all the syncytiotrophoblast (Figure 4,D and 4E), the villous stromal cells (Figure 4F, arrow), and the extravillous endovascular trophoblast cells (Figure 4G), the fetal origin of these cells were indicated by the anti-cytokeratin antibody staining (Figure 4 G inset, brown), and the vascular wall was stained by anti-actin antibody (red). The pattern of Bcl-2 expression in the syncytiotrophoblast was similar to that of the apoptotic nuclei distribution (Figure 2). In the maternal compartment, Bcl-2 could be detected in some stromal cells (Figure 4H). Notably, the cytotrophoblasts lining the villi, within the cell columns, and the glandular epithelia were negative for Bcl-2 staining. The semi-quantitative expression level of Bcl-2 in different cell types at the various stages was summarized in Table 1. A gradual increase of Bcl-2 staining was observed in the syncytiotrophoblast as gestation advances.

Figure 4
figure 4

Immunohistochemical staining for Bcl-2 at implantation sites of the rhesus monkey. Bcl-2 staining is red, and nuclear counterstain blue. A, villous plancenta on D17. B, villous plancenta on D19. C, extravillous trophoblast cells in the basal plate of D17. D, villous plancenta on D28. E, villous plancenta on D34. F, villous plancenta on D34 under a higher magnification. G, the extravillous endovascular trophoblast cells; in the inset, the fetal origin of these cells was confirmed by anti-cytokeratin antibody (brown), and their position within the vascular wall was confirmed by anti-actin antibody staining (red). H, decidua. I, negative control. Vi, placental villi. ST, syncytiotrophoblast. CT, cytotrophoblast. Sc, stromal cells. Ge, glandular epithelium. Evc, extravillous cytotrophoblast. Scale bars represent 50 μm.

Full size image
Table 1 Semi-quantitative assessment of the immunohistochemical staining of Bcl-2 in the placenta of rhesus monkey.
Full size table

P53 expression in implantation site at early pregnancy

The expression profile of P53 was also acquired by using the immunohistochemistry. On D17 and D19, the expression of P53 was only confined to a small number of nuclei in the syncytiotrophoblast (Figure 5,A,B). On D28 and D34, its expression was observed not only in the syncytiotrophoblast (Figure 5C,D) but also in the nuclei of cytotrophoblasts lining the villi (Figure 5E) and within proximal tip of cell columns (Figure 5F) where a proliferative activity was high as indicated by Ki67 staining (Figure 3). Clustered P53-positive nuclei were seen in the syncytiotrophoblast covering the basal feet of the anchoring villi (Figure 5G), coincident well with the strong apoptosis detected by TUNEL (Figure 2E). P53 was also expressed in some stromal cells (Figure 5H) of the uterine endometrium.

Figure 5
figure 5

Immunohistochemical staining for P53 at implantation sites of the rhesus monkey on D17 (A), D19 (B), D28 (C, H), and D34 (D, E, F, G) of gestation. P53 was stained dark in nuclei. A-D were villous placenta under a lower magnification. The inset of panel A shows the staining in the syncytiotrophoblast covering the basal feet of the anchoring villi under a higher magnification. E, staining in villous placenta under a higher magnification. F, staining in cell columns. G, syncytiotrophoblast covering the basal feet of the anchoring villi under a higher magnification. H, the endometrium with arrows indicating stromal cells. ST, syncytiotrophoblast. CT, cytotrophoblast. Scale bars represent 50 μm.

Full size image

Discussion

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 [1]. 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.

In our previous study, Bax expression was found at the Fetal-Maternal Interface of Rhesus Monkey [17]. Bax is a Bcl-2 family member that promotes cell death susceptibility, possibly by countering the effect of Bcl-2 on cell survival through heterodimer interaction. Bax to Bcl-2 "rheostat" may be a critical factor in regulating apoptosis in multiplicate tissues. As shown in Figure 6, Bax was found expressed in the placenta and glandular epithelium of endometrium and all kinds of cells in placental villi, and no obvious change was observed between different time points from D17 to D34 in placental villi. Therefore, we speculated that Bcl-2 may play a more important role on controlling the apoptosis in placental villi. The diffusive expression of Bcl-2 in syncytiotrophoblast obtained from the first trimester human placenta has been reported recently [7, 23–25]. Our observation on the Bcl-2 expression in syncytiotrophoblast at later stages (D28-D34) agreed well with these data. As shown in this study, although Bcl-2 was expressed, apoptotic nuclei still exsisted in the same region. This phenomenon implies that the expression of Bcl-2 is not sufficient to completely inhibit the apoptosis in the syncytiotrophoblast. Therefore, the role of Bcl-2 here becomes an interesting question. Multiple nuclei sharing the same cytoplasm is a morphological characteristic of syncytiotrophoblast. In such cells, the apoptotic signal may be transmitted from one nuclear to another, and cause a spontaneous abortion. Therefore, the number of nuclei undergoing apoptosis in the syncytiotrophoblast should be limited by some mechanism in order to ensure normal embryo development in normal pregnancy [1]. We speculate that Bcl-2 may be included in this mechanism. The major role of apoptosis-associated Bcl-2 expression in the syncytiotrophoblast might be to limit the nuclear degradation to a special area and inhibit the spread of cell apoptosis signals to the other nuclei sharing the same cytoplasm, thus sustain cell survival in these multi-nucleated cells. Toki et al has also suggested that Bcl-2 might play a major role in avoiding the possible excessive nuclear degradation in syncytiotrophoblast [26]. Further studies, however, are needed to prove this speculation. The immunostaining for Bcl-2 was also detected in part of the extravillous and endovascular cytotrophoblast in our study. These subtypes of cytotrophoblast lost the capacity of proliferation (Ki-67-negative), but they did not undergo apoptosis (negative in TUNEL assay). Therefore, we hypothesize that Bcl-2 may also participate in regulation of the extravillous trophoblast apoptosis by stimulating the cellular survival.

Figure 6
figure 6

Immunohistochemical staining for Bax at implantation sites of the rhesus monkey on D28. Bax staining is brown, and nuclear counterstain blue. A, villous plancenta, positive staining was found in all the cells. B, endometrium, glandular epithelium was positive for Bax staining. Vi, placental villi. Ge, glandular epithelium.

Full size image

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 [27]. 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 [8]. 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 [29]. 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.