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Endopolyploid and proliferating trophoblast cells express different patterns of intracellular cytokeratin and glycogen localization in the rat placenta
Tatiana G. Zybina, Grigori I. Stein and Eugenia V. Zybina1
Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia
The presence of keratin intermediate filaments is a characteristic of trophoblast differentiation. Meantime, their intracellular localization in the functionally different subtypes of placental trophoblast is poorly investigated in rodent, whereas their placentae are being broadly investigated in recent years as a model of the feto-maternal interaction. The purpose was to study the intracellular distribution of cytokeratin filaments in correlation with glycogen deposits, both being important constituents of the trophoblast cells in rat placenta. Different rat trophoblast cell populations exhibited different patterns of cytokeratin immunolocalization. The most intensive immunostaining was observed in the highly endopolyploid SGTCs (secondary giant trophoblast cells) at the border with decidua basalis. The most prominent cytokeratin-positive threads were found at the periphery of cytoplasm and in the extensive system of cytoplasmic sprouts by which the SGTC connect each other. Similar cytokeratin intensity and distribution was detected in the TSC (trabecular spongiotrophoblast cells) of the junctional zone of placenta that line the lacunae with the maternal blood. Clusters of highly proliferative pre-glycogen as well as glycogen cells showed some weaker cytokeratin signals mostly in the perinuclear and peripheral zones of cytoplasm. At the 11.5th to the 13.5th day of gestation, the interstitial and endovascular invasive endopolyploid TGTCs (tertiary giant trophoblast cells) prove the intensive cytokeratin staining throughout the cytoplasm and its sprouts. Meantime, the TGTCs were glycogen negative. By contrast, glycogen was heavily accumulated in the glycogen cells that belong both to the junctional zone of placenta and the cuff of the central arterial channel underlying the monolayer of endovascularly invading TGTCs. Thus, the TGTCs that are first to penetrate into the depth of the uterine wall do not contain glycogen but are accompanied by the glycogen-rich cells. The SGTC also contained the prominent deposits of glycogen at the periphery of cytoplasm and in the cytoplasmic sprouts. At the 16th day of gestation, an extensive interstitial invasion of the cytokeratin-positive glycogen trophoblast cells from the junctional zone was observed. The patterns of cytokeratin and glycogen intracellular localization are specific for each subtype of the rat trophoblast; that is, most probably, accounted for by the functional diversity of different trophoblast populations, i.e. patterns of invasion/phagocytosis and their involvement in a barrier at the feto–maternal interface.
Key words: cytokeratin, intermediate filament, placenta, rat, trophoblast
Abbreviations: dpc, day post coitum, JZ, junctional zone of the placenta, PGTC, primary giant trophoblast cell, SGTC, secondary giant trophoblast cell, TGTC, tertiary giant trophoblast cell, TSC, trabecular spongiotrophoblast cell
1To whom correspondence should be addressed (email firstname.lastname@example.org).
In rodent, the trophoblast invasion that ensures the embryo implantation and its anchoring on the uterine wall includes several steps. At these steps, the trophoblast cells differentiate into a range of cell types able to invade different zones of the uterine wall; they differ by the period and mode of invasion, in particular, by their depth of penetration into the uterine wall.
In rodents, the primary and secondary giant trophoblast cells are first to invade the uterine wall during the embryo implantation. The PGTC (primary giant trophoblast cell) lyse the uterine epithelium and migrate antimesometrially (Al-Abbas and Schultz, 1966). In rat, the SGTCs (secondary giant trophoblast cells) phagocytose the epithelial lining of the implantational camera, decidual and maternal blood cells (Orsini, 1954; Jollie, 1965; Tachi et al., 1970; Zybina, 1976a, 1976b; 1986; Welsh and Enders, 1985; Bevilacqua and Abrahamson, 1989, 1991; Zybina and Zybina, 2005). Migration of SGTC is achieved without loss of contact between trophoblast cells; thereby, they form a continuous ‘front of invasion’ (Orsini, 1954; Dickson and Bulmer, 1960; Zybina and Zybina, 2005) that subsequently produces a barrier between the maternal and fetal parts of the placenta.
Unlikely, the PGTC and SGTC, the TGTCs (tertiary giant trophoblast cells) in rat and mouse (Zybina et al., 2000; Zybina and Zybina, 2005) or endovascular and interstitial giant trophoblast cells (Adamson et al., 2002; Ain et al., 2002; Caluwaerts et al., 2005) become immersed into the allogenic maternal tissues to a greater degree.
The presence of keratin intermediate filaments is characteristic of trophoblast differentiation. Meantime, the intracellular localization of cytokeratins in the functionally different subtypes of placental trophoblast is poorly investigated, whereas their placenta is largely investigated for recent years as a model of the feto-maternal interaction (Redline et al., 1993; Teesalu et al., 1999; Georgiades et al., 2002; Coan et al., 2006). It seemed interesting to study the intracellular distribution of cytokeratin in correlation with the glycogen deposits, both of them being important constituents of the trophoblast cells in the rat placenta.
In this regard, intracellular localization of cytokeratin in different pathways of trophoblast invasion appears to be of special interest. One of the main invasion pathways in rodent trophoblast is accomplished by the glycogen cells (Bridgman, 1948; Peel, 1989; Ain et al., 2002; Vercruysse et al., 2006, Konno et al., 2007). Meantime, we did not observe a uniquely determined and unambiguous correlation between trophoblast invasiveness and glycogen storage (Zybina and Zybina, 2005). That is why we made a detailed study in order to clarify the modes, depth and time of invasion of glycogen-containing trophoblast cells and to compare glycogen positivity of different trophoblast cell populations. Besides, we tried to find a connection of the glycogen and cytokeratin localization, the latter being also bound with their invasive activity and is a marker of trophoblast cells in the invaded stroma. It is also of interest to evaluate glycogen localization as a marker of different trophoblast cell populations. Therefore, the aim of this work was to study the intracellular distribution of cytokeratin in correlation with glycogen deposits in different trophoblast cell populations in the rat placenta.
2. Materials and methods
Animal experimentation was carried out according to the guidelines of the Animal Use Committee of the Institute of Cytology RAS.
Placentae of white random-bred rats (Rattus norvegicus) at the 11.5th, 13.5th, 16.5th, 18.5th and 19th dpc (day post coitum) were fixed in ethanol–acetic acid (3:1), paraffin-embedded, and the 7-μm sections were cut. Immunohistochemical testing using the cytokeratin pan antibodies was carried out according to the slightly modified standard procedure (Mühlhauser et al., 1993). The monoclonal anti-human antibodies cytokeratin pan were used as the primary antibodies. The deparaffinized sections were incubated with bromelin for 15 min at 37°C. Endogenous peroxidase activity was quenched by the 15-min incubation with 3% hydrogen peroxide. Non-specific antibody binding was blocked by incubation for 30 min in rabbit serum, then the sections were incubated with the primary antibodies diluted 1:300 in 0.6% Tris buffer, 1.5% BSA (pH 7.6), then for 30 min with biotinylated rabbit anti-mouse antibodies (1:400) and with streptavidin–peroxidase (1:400); 3′-3-diaminobenzidine was used as a substrate for peroxidase.
PAS (periodic acid–Schiff reagent) reaction was performed in order to estimate glycogen localization. The slides were incubated in 0.8% potassium periodate, 0.23% HNO3 for 1.5 h at room temperature, held in the tap water for 5 min and rinsed in aqua dest. Then, the slides were placed into Shiff reagent for 1.5 h at room temperature. After that, the slides were rinsed in aqua dest and incubated in three portions of 0.5% Na2S2O3, 0.05-N HCl for 3 min each. Then, the slides were dehydrated in a series of alcohols and embedded into Canada balsam. The slides were examined under the photomicroscope Axiophot (Carl Zeiss) with the objectives 10/0.30, 20/0.50 and 40/0.50. The photos were taken using the CCD Camera Coolpix-500 (Nikon), image size 2560×1520.
3.1. Cytokeratin immunostaining
Different rat trophoblast cell populations showed different patterns of cytokeratin immunolocalization. The most intensive immunostaining was observed in the highly endopolyploid SGTCs at the border with decidua basalis (Figure 1a). As early as at the 11.5 dpc, the most prominent cytokeratin-positive threads were found at the periphery of the cytoplasm and in the extensive system of cytoplasmic sprouts by which the SGTCs connect each other (Figures 1a, 1b, 1d, 1e). The cytokeratin filaments seem to form a kind of a framework of the SGTC layer that probably serves as a barrier between the fetal and maternal parts of the placenta.
Simultaneously, some of the SGTCs protrude the decidual tissue by means of their large nipper-like, highly cytokeratin-positive sprouts that surround the wide accumulations of decidual cells (Figure 1e), the latter, probably, undergoing subsequent degradation. This process seems to take place during the lengthy period of pregnancy studied here (Figures 2a, 2b); it is the most strongly pronounced in a part of the SGTC layer near the central arterial channel. Therefore, the SGTCs persist in their intrauterine invasion within a continuous ‘front’ of trophoblast cells tightly connected to each other. Such a ‘front’ is interrupted in the centre of the placental disc where the central (maternal) arterial channel traverses the placenta.
As early as at the 11.5 dpc, the trophoblast cells of the JZ (junctional zone of placenta) begin to differentiate into glycogen cells, and the spongiotrophoblast cells that form trabecules lining the lacunae with maternal blood (Figure 1d). These TSCs (trabecular spongiotrophoblast cells) have larger size and long cytoplasmic outgrowths that contact the neighbour trabecular cells, thereby forming almost the continuous layer around the maternal blood lacunae. In the TSCs, a similar cytokeratin intensity and distribution was detected compared with SGTC. Cytokeratin is localized throughout the cytoplasm, and especially prominent immunostaining is also observed at the periphery of the cells and their sprouts. The cytokeratin filaments also seem to form a framework (Figure 1d). The rest of the trophoblast cells of the JZ zone that include the highly proliferative and/or differentiating (‘pre-glycogen’) cells show a weaker cytokeratin staining mostly throughout the cytoplasm; a somewhat stronger signal is observed in the perinuclear zones (Figure 1d). These cells, most probably, subsequently differentiate into glycogen cells as well as new generation of TSCs.
At the 13.5 dpc, the cytokeratin immunostaining in the SGTC and the JZ proves to be similar to the 11.5 dpc (Figure 2a). Beginning from the 13.5 dpc, clusters of glycogen cells embedded into the network of TSCs showed a clearly weaker cytokeratin signal located mostly in the peripheral and perinuclear zones of the cytoplasm that, probably, may be accounted for by the heavy glycogen deposits in these cells (see the Discussion).
All the more profound differences are observed at the 16.5 dpc. In the glycogen cells, the cytokeratin is mainly located in the perinuclear zone. The TSCs that delimit the maternal blood lacunae retain their immunostaining throughout the cytoplasm and its sprouts (Figure 2c).
Besides the SGTC invasion, a deeper trophoblast invasion was observed beginning from the 11.5 dpc in the area of the central arterial channel. In this region, the smaller cytokeratin-positive trophoblast cells penetrate interstitially into the decidualized endometrium (Figures 1c, 1e). Very often, they seem to connect the invading SGTCs via their cytokeratin-positive sprouts.
By the 16.5 dpc, an extensive interstitial trophoblast invasion is observed in a region of the mesometrial triangle. The invasion is accomplished by both the intensively stained spindle-shaped cells and, most probably, by the glycogen cells as was observed by other authors (Vercruysse et al., 2006).
3.2. Glycogen deposits
Characteristics of the glycogen deposits of placenta changed in the course of differentiation of the JZ and in the course of invasion. At the 11.5 dpc, all the trophoblast cells in JZ showed rather uniform intensive PAS reaction (Figures 3a, 3b). A significant differentiation is observed beginning from the 13.5 dpc (Figures 3c–3e): the spongiotrophoblast cells that surround clusters of glycogen cells show a very weak PAS reaction, whereas the glycogen cells show the prominent glycogen deposits.
Nevertheless, as early as at the 11.5 dpc, a small population of JZ cells, probably precursors of the TGTCs (Orsini, 1954) or endovascular cells, appears in the zone of the central arterial channel where a gate in the SGTC layer is observed. The accumulations of the trophoblast cells of JZ lose their PAS positivity (Figure 3a). Beginning from the 13.5 dpc, these cells are seen to migrate along the central arterial channel forming its unilaminar lining (Figures 3d, 3e). Simultaneously, a highly PAS-positive part of glycogen cell accumulations migrate into the decidua basalis in a form of multilaminar stratum that underlies TGTC and form the outer ‘cuff’ of the central arterial channel (Figure 3e). Meantime, at the 13.5 dpc, the glycogen cell stratum does not migrate to the significant depth of the decidualized endometrium, whereas TGTC continue their endovascular migration towards the myometrium (Figure 3d).
Thus, at the site where the central arterial channel traverses the border of the fetal and maternal part of placenta, a thick, partly multilayered muff of glycogen-rich cell underlies a monolayer of TGTCs lining the lumen of the channel. The ‘muff’ consists of the stratum of glycogen cells at the feto–maternal interface and the proximal decidua, on one hand, and the granulated metrial gland cells [i.e. uterine NK (natural killer) cells] at the distal part of the decidua and myometrium, on the other hand (Zybina et al., 2000; Zybina and Zybina, 2005).
At the 16.5–18.5 dpc, the glycogen cells retain their glycogen deposits, whereas more massive layers of TSC cells have very weak PAS reaction. At this time, the glycogen cells appear to migrate mesometrially in the zone around the central arterial channel and then spread out interstitially throughout the mesometrial triangle (Vercruysse et al., 2006). The glycogen cell differentiation is confirmed by the Methyl Green–Pyronin staining data. The TGTCs that are first to migrate into the depth of the decidua show more intensive Pyronin staining (Figure 4a). The glycogen cells that follow the TGTCs have the same pyroninophilia, but in the course of their differentiation, they lose their Pyronin staining by the moment of their extensive interstitial migration (Figure 4b).
From the beginning of implantation, the trophoblast cells invading the uterine wall do not lose, as a rule, their intercellular connections. Thus, the ectoplacental cone cells move in a mesometrial direction in a form of a continuous stratum, and subsequently, i.e. beginning from the 11.5 day of gestation, form the SGTCs that represent an indivisible ‘front’ of invasion in the developing placenta (Bridgman, 1948; Orsini, 1954; Dickson and Bulmer, 1960; Zybina and Zybina, 2005).
Over a prolonged period of pregnancy, the layer of SGTC at the border with decidua basalis, most probably, represents a barrier between the semiallogenic maternal and fetal parts of placenta. The clear-cut peripheral localization of cytokeratin intermediate filaments seems to promote continuity of the SGTC layer in which the cells are bound to form a framework that delimit the underlying layers of the low-polyploid proliferative and differentiating trophoblast cells from the decidua basalis. It cannot be ruled out that the cytokeratin filaments ensure continuity of the ‘barrier’ SGTC layer. It is supported by the data on the targeted deletion of keratins 18 and 19 that leads to trophoblast fragility and embryonic lethality (Hesse et al., 2000). In the knockout experiments, mice double deficient for K18 and K19 showed complete loss of keratin filaments that resulted in cytolysis of the trophoblast giant cells followed by haematomas in the trophoblast layer.
According to the data obtained in this study, SGTC invasion persists until the latest stages of pregnancy. The peculiarity of invasion at this period appears to be the penetration of cytoplasmic ‘tongues’ of one or some neighbour SGTCs inside the decidua and encompassing great areas of the tissue that probably results in its progressive degradation. The cytokeratin filaments that show an intensive immunolabelling in the cytoplasmic sprouts seem to take part in this process. Thus, the peripheral cytokeratin filaments seem to allow SGTC to combine phagocytosis of decidual cells and invasion on one hand and maintenance of the continuous barrier that probably protect the maternal and fetal organisms from mutual immunological attacks.
Endopolyploidization is, most probably, of great importance for the SGTC invasive and protective functions. Indeed, the fast growth of SGTC population may be achieved due to endoreduplication that does not imply a significant rearrangement of intracellular structure that is characteristic of mitosis. As a consequence, peripheral intermediate filaments that form a framework of tightly attached SGTC may stay intact during the significant part of pregnancy. Besides, the giant size of SGTCs makes encompassing the large accumulation of decidual cells by enormous SGTC sprouts possible that probably results in the progressive lysis and/or phagocytosis of decidual tissue.
Denker (1993) indicates that one of the biological paradoxes is that the blastocyst trophoblast, when becoming invasive, loses a part of its typical epithelial organization. Meantime, the trophoblast cells do not lose their connections on the lateral membrane, so they migrate as sheets rather than as individual cells. It should be mentioned that such a way of cell migration is also characteristic of the typical epithelial cells, for example, in keratinocyte stratum in the case of wound healing (Schmidt and Friedl, 2010).
The TSCs of JZ, according to our observation, show the similar cytokeratin localization: threads of peripheral filaments that continue into processes by which these cells are closely attached to each other thereby encompassing the maternal blood lacunae. It cannot be ruled out that the layer of closely connected TSCs delimit the underlying clusters of the actively proliferating trophoblast cells and differentiating glycogen cells from the allogenic maternal blood. In this respect, the trophospongial cells are structurally and functionally similar to the SGTCs. It seems to be of importance that these cells show continuity with the SGTC stratum, both cell populations probably representing a whole system that separate embryo from the maternal tissues. In this connection, the data on compound mutants on Cytokeratin 8 and 19 should be mentioned (Tamai et al., 2000). According to these data, inactivation of both K19 and K8 results in an excessive number of giant trophoblast cells, whereas the embryos lacked proper labyrinthine trophoblast and junctional zone development. The giant trophoblast cells were pulled apart but not tightly attached to each other. The number of the labyrinthine trophoblast and spongiotrophoblast cells was decreased in cell number, and they were poorly organized. This apparently caused flooding of maternal blood directly into the fetal tissue, where these trophoblast cells normally separate embryonic blood from maternal circulation. As a result, most probably, there occurred an intermixing of the maternal and the fetal blood in the placenta due to inability to keep the circulation system separate (Tamai et al., 2000).
It is conceivable that mesenchymal and epithelial features are characteristic of another type of trophoblast invasion, i.e. endovascular one. In this case, also a close connection of cytokeratin-positive trophoblast cells forming ‘chains’ migrating along the uteroplacental arteries, against the blood flow, is observed.
Glycogen cells represent another type of trophoblast cells migrating into the depth of the endometrium. From the beginning of their differentiation at the 13.5 day of gestation, they differ from other trophoblast cells by cytokeratin localization. Immunolabelling is often observed in a form of a thin rim around the nucleus and at the periphery cytoplasm. It is quite possible that such a localization of cytokeratin filaments is connected with glycogen accumulation in the cytoplasm. The pattern of their invasive behaviour differs from the above-mentioned trophoblast cell populations. First, they accompany the tertiary (endovascular) trophoblast cells in the zone of ‘gate’ in the SGTC layer where the central arterial channel traverse the fetal and maternal parts of the placenta. At the beginning of their differentiation, the glycogen cells are capable of ‘shallow’ invasion into endometrium around the central arterial channel thereby forming a mutilayered ‘muff’ underlying a layer of the TGTCs. At this time (13.5 day of gestation), these cells stay tightly attached to each other just like the clusters of glycogen cells within the junctional zone of placenta.
Glycogen cells are considered to be among the main subjects of the interstitial invasion (Bridgman, 1948; Peel, 1989; Adamson et al., 2002; Caluwaerts et al., 2005; Vercruysse et al., 2006; Coan et al., 2006) though other subtypes of trophoblast cells may take part in this process. These subtypes include the TGTCs (Zybina and Zybina, 2000; 2005) as well as ‘elongated’ trophoblast cells that invade decidua at the late stages of pregnancy (Vercruysse et al., 2006). According to our observation, the interstitial invasion starts as early as at the 11.5 day of gestation. Other authors describe the interstitially invading trophoblast cells in rat beginning from the 13.5 day of gestation (Konno et al., 2007). Interestingly, these cells are often bound into ‘chains’, but they can also migrate in a form of single spindle-like cells. Cytokeratin filament localization throughout the cytoplasm and its sprouts may, probably, play a role both in their movement and in maintenance of their contacts to each other.
Beginning from the 16.5 day of gestation, the character of glycogen cell invasion changes: they, probably, lose their contacts with each other and rush into the depth of decidua in a form of a vast stream through the ‘gap’ in the SGTC layer around the central arterial channel, as also described by Vercruysse et al. (2006). Beginning from this time, an extensive interstitial dissemination of glycogen cells and spindle-like cells throughout the whole mesometrial triangle is observed (Caluwaerts et al., 2005; Vercruysse et al., 2006). This process in rat placenta is observed until the 20th day of gestation. Therefore, it suggests that the trophoblast cells that do not have a system of cytokeratin filament at the periphery of cytoplasm possess alternative invasive properties and, possibly, are more capable of interstitial migration in a form of single cells.
It seems to be of interest that in the course of formation of the JZ, differentiation of the strongly cytokeratin-immunopositive PAS-negative TSC and PAS-positive glycogen cells with weak cytokeratin immunostaining is observed. A similar tendency is observed in the relationship between cytokeratin- and glycogen-rich cells in the zone of endovascular trophoblast invasion. The glycogen-free endovascular (tertiary) giant trophoblast cells are underlain by the ‘muff’ of glycogen-rich trophoblast cells that, in the depth of the uterine wall, give the way to the ‘cuff’ of uterine NK cells containing prominent glycogen granules. Up to now, It is not clear why the deeply invading TGTCs are glycogen negative; meantime, they are followed by the glycogen-rich cells. Within the fetal part of the placenta, the TSCs with a weak PAS reaction also come in contact with maternal blood, the cells also being underlain by glycogen-rich cambial and glycogen trophoblast cells. It cannot be ruled out that such a relationship plays a role in the formation of a barrier between the genetically foreign fetal and maternal organisms.
Interestingly, an extensive interstitial invasion of glycogen cells is observed by the time of progressive depleting of glycogen granules from the glycogen cells at 18–20 dpc. It may be accounted for by the start of glycogen deposition in the rat embryonic liver (Kudryavtseva, 1967). It is conceivable that at this moment, the glycogen cells lose their function of embryonic glycogen storage. It cannot be ruled out that in this case, also the cells devoid of glycogen are capable of the active invasion in the depth of semiallogenic tissues.
Tatiana Zybina contributed to the investigation project design, performed the immunohistochemistry reactions, examined the slides and analysed the data. She also drafted the manuscript and arranged the Figures. Grigory Stain arranged and performed works at the photomicroscope and contributed to the data analysis. Eugenia Zybina contributed to the investigation project design, collected the specimens of placentas, performed the PAS and Methyl Green/Pyronin cytochemistry reactions, examined the slides, analysed the data and drafted the manuscript.
We thank Professor L.Z. Pevzner for the help in translating and editing the manuscript.
This work was supported by the Governmental Program for the study of Molecular and Cell Biology.
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Received 23 April 2010/13 November 2010; accepted 7 February 2011
Published as Cell Biology International Immediate Publication 7 February 2011, doi:10.1042/CBI20100278
© The Author(s) Journal compilation © 2011 Portland Press Limited
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 1 Localization of cytokeratin in different trophoblast cell populations of rat placenta at 11.5 dpc
Figure 2 Cytokeratin immunostaining of trophoblast cells in the rat placenta at the 13.5 (a) and 16.5 (b, c) dpc
Figure 3 Glycogen deposits in the trophoblast cells in rat placenta at 11.5 (a, b) and 13.5 (c–e) dpc