Biology and Medicine
Editor-in-Chief: Eytan R. Barnea MD, FACOG
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EARLY
PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG |
Key words: invasion, proliferation, reproduction, genome, placenta, rodents
Correspondence: Dr.T.G. Zybina, Laboratory of Cell Pathology, Institute of Cytology RAS, 4 Tikhoretsky Ave., St.Petersburg 194064, Russia, Tel. 7(812) 247-1859, Fax: 7(812) 247-0341, e-mail:zybina@mail.cytspb.rssi.ru
Acknowledgements: The authors are grateful to Dr. L.Z.Pevzner for translating
and editing the manuscript. The study was supported by the Russian Foundation for Basic
Investigation
(grant N98-04-48322).
Abstract
Using 3H-thymidine labeling and cytophotometric DNA content measurement in nuclei and mitotic figures, proliferative activity and genome reproduction peculiarities have been studied in highly invasive, primary and secondary giant trophoblast cells (pGTCs and sGTCs, respectively) as well as in the low invasive junctional zone and labyrinth trophoblast cells (JTCs and LTCs) of rat placenta. It has been shown that different extent and patterns of phagocytic activity of pGTCs and sGTCs correspond to different developmental stages. An inverse relationship has been observed between capability for mitoses and invasive and phagocytic activities. The pGTCs and sGTCs lose their mitotic activity from the start of their differentiation. Nevertheless, they continue reproduction of their genome and undergo a series of endoreduplication cycles to reach the ploidy degree of 256-1024c. In contrast, the JTCs and LTCs have, as a rule, no invasive and phagocytic activities, while preserving proliferative properties up to 15 day of gestation. They undergo initial polyploidization via uncompleted polyploidizing mitoses up to octaploid level and then pass to the endoreduplication cycle that excludes mitoses. Such a way of cell reproduction has been suggested to play a protective role, as it rules out contacts of the trophoblast cell genome with chromosomes of the phagocyted allogenic maternal tissue.
Introduction In studying phenomenon of embryo implantation and placentation, a great attention has been paid by many authors to a number of specific features and unique properties of trophoblast cells, i.e. their capability of invading, and migrating deeply into, maternal tissues (Orsini, 1954, Zybina and Tikhomirova, 1963, Enders and Schlafke, 1969, 1971, Tachi et al., 1970, Smith and Wilson, 1974, Pijnenborg et al., 1974, 1981, Bevilacqua and Abrahamson, 1988, Blankenship et al., 1992, Damsky et al., 1993, Lala, 1997).For many years, although to a lesser extent, an attention has been drawn to the ability of trophoblast cells to phagocytose endometrial cells (Maximov, 1900, Zybina and Tikhomirova, 1963, Zybina, 1986, Zybina and Zybina, 1996, Jollie, 1981, Walsh and Enders, 1987, Bevilacqua and Abrahamsom, 1988, Aplin, 1991, Hoffman, Wooding, 1993, Albieri and Bevilacqua, 1996). Phagocytosis can be considered the most ancient cellular protective mechanism (Mechnikov, 1951); to some extent, it is involved in all kinds of immunological reactions. Therefore, the trophoblast cell capability for lysis and phagocytosis seems to be very important for embryo penetration into allogenic maternal tissues. Of great significance is protective role of trophoblast cells directed against maternal alloimmune attack (Kolb et al., 1984, Goldsobel et al., 1986, Lala et al, 1988, Stewart and Mukhtar, 1988, Mikhailov et al., 1994, Lin et al., 1993); this protection can be facilitated by different mechanisms including phagocytosis.
Many trophoblast cell populations are known to be polyploid (for review, see Zybina and Zybina, 1996). However, we believe it to be of interest to examine specifically the relationship between invasive and phagocytic properties of trophoblast cells with respect to their capability or incapability for proliferation and other ways of genome reproduction under different conditions of interaction with allogenic maternal tissues. The purpose of the present study was to examine capability of different trophoblast cell populations for chromosome reproduction under different conditions of their contact with maternal tissues, i.e. in the course of their invasion into endometrium and stationary location at the boundary with the endometrium and, in case of lack of close contact, with endometrial tissues.
Methods
This study used a combination of morphological observation of different trophoblast cell populations in rat placenta with historadiographical identification of DNA-synthesizing nuclei with 3H-thymidine labeling as well as cytophotometric determination of DNA content in nuclei and mitotic figures. AutoradiographyTo measure DNA content, squash preparations were prepared from rat placentas at 12, 13 and 14 dpc. Pieces of placenta were fixed in the ethanol-glacial acetic acid mixture (3:1), dehydrated in 96% ethanol, macerated in 45% acetic acid and placed on dry ice (CO2). Cover glasses were removed from the frozen preparations, and these were dried and stained with Feulgen stain (hydrolysis in 5N HCl for 30 min at room temperature). As a standard for the haploid and diploid DNA amount, smears of sperm cells and peripheral blood lymphocytes as well as embryonal nuclear erythrocytes were used. The cytophotometric determination of the DNA content was performed using a Morphoquant automated image analyzer (Carl Zeiss, Jena, Germany) composed of a scanning microscope and computer (wavelength 575 nm, objective 50x ). The total of 900 nuclei of sGTCs and of 200 nuclei at each developmental stage in JTCs and LTCs were analyzed. To determine ploidy levels of nuclei and mitotic figures in the trophoblast cells as well as of nuclei in spermatozoa, lymphocytes, and fetal red blood cells, histograms were constructed of distribution of the DNA content in logarithmic scale (abscissa: decimal logarithm of the integral optical density that reflects the DNA content in arbitrary units, ordinate: the number of nuclei or mitotic figures). The modal value of the DNA content in lymphocytes and fetal red blood cells was taken as the diploid DNA content (2c), while the modal value in nuclei of spermatozoa, as the haploid DNA content (1c). Based on these values, the theoretically expected mean values of the DNA content in the nuclei were calculated: 4c, 8c, 16c, 32c, etc. Mitotic figures with the DNA content 4c were considered mitoses of the diploid cell. Accordingly, mitoses with the DNA contents 8c and 16c were considered those of the tetra- and octaploid cell, respectively.
Results Primary and secondary giant trophoblast cellsBy 10 dpc, the relationship between invasiveness of pGTCs and their capability for phagocytosis changed noticeably: they lost their motility and formed a stationary stratum at the boundary with the decidual tissue. At that time they could phagocytose mainly maternal blood cells (Figure 1C-E). By this stage of placenta formation, they seemed to lose their functional significance and degenerated.
From the start of active phagocytosis or, perhaps, even somewhat earlier, pGTCs were not capable of dividing mitotically. Nevertheless, their nuclei took up actively 3H-thymidine, which suggests a switch to the endoreduplication cycle. As a result, they reached as high level of ploidy as 256c (Zybina, 1986).
The secondary giant trophoblast cells (sGTCs) appeared from the polar trophoblast at the top of ectoplacental cone (EC). Beginning from 7 dpc, the EC cells moved mesometrially, and the peripherally located sGTCs lysed and phagocytosed the IC epithelial lining, so that by 10 dpc they were attached directly to the decidua basalis.
The growing sGTCs moved deeply inside decidua basalis via lacunae (Figure 2B) to lyse decidual and endothelial cells and to form an extensive zone of migration. At this stage, a great number of phagocyted erythrocytes were observed in the cytoplasm of sGTCs. Groups of several sGTCs surrounded large zones of decidual tissue by means of cytoplasmic projections that penetrated beneath endothelium (Figure 2B) and through the extracellular matrix. These sGTC groups seemed to lyse pieces of decidual tissue (Figure 2C); this is the so-called «group phagocytosis» (Dondua, 1954).
After 14 dpc, both invasive and phagocytic activity of sGTCs decreased significantly. The sGTCs were tightly attached to each other and formed an almost continuous layer at the periphery of the fetal part of placenta (Figure 2A). In several limited sites, a group phagocytosis of the decidual tissue was preserved (Figure 2A).
Since this stage, a part of sGTCs underwent progressive degeneration, probably via apoptosis that involved most sGTCs by 18 dpc, a part of them remaining vital, as a rule, almost until the end of pregnancy. In most sGTCs, nuclei became fragmented, which occurred most commonly not long before degeneration (Zybina and Zybina, 1996).
At the 14 dpc, most sGTC nuclei were shown historadiographically to incorporate 3H-thymidine, whereas at the 18 dpc, only occasional nuclei were labeled. Therefore, sGTC were not capable for mitotic proliferation, however, they did not stop replicating DNA.
Cytophotometry of DNA content in the sGTC nuclei showed intensive sGTC polyploidization during the period of invasion. By 12 dpc, most sGTC nuclei reached 128-256c nuclei prevailed (Figure 3). Such a high level of ploidy was achieved on full arrest of mitoses, i.e. via endoreduplication (Figure 4). Subsequently, at the 15-18 dpc, the genome multiplication continued to result in 256-512c, a small part of the nuclei reaching 1024c. Therefore, a conclusion can be made that processes of invasion and fast polyploidization coincide. Nonetheless, sGTCs reached the highest level of ploidy, 256-1024c, at a period of their more stationary presence inside the continuous sGTC layer that forms a barrier between the fetal and maternal zones of placenta.
Junctional zone and labyrinth trophoblast cellsJTCs can divide mitotically until 15 dpc, whereas labyrinth trophoblast cells (LTCs), until 17 dpc. The maximum of mitotic activity (10%) in JTCs was observed at 13-14 dpc, then by 15 dpc it fell to 1%, and in a day no mitoses were revealed at all.
Nuclei of JTCs were found to take up 3H-thymidine during 3 days after cessation of mitoses. Therefore, chromosome reproduction continued without mitotic divisions. The DNA cytophotometry has shown that at the period of the highest mitotic activity (13 dpc), nuclei 2c (39%) and 4c (46%) prevail in JTCs, the higher levels of ploidy (8c, 16c, and 32c) occurring much less often (9%, 5%, and 1%, correspondingly). By 14 dpc, a part of diploid nuclei passed to 4c, the percentage of 8c rising. After cessation of mitotic activity, the genome multiplication continued. Thereby by 18 dpc, most nuclei reached 16c (52%), and a significant percentage of nuclei (16%) corresponded to 32c, whereas the 4c and 8c nuclei accounted for only 9% and 23%, respectively. Judging by nuclear sizes, the cells of trophospongium had higher ploidy levels than the glycogen cells.
The so-called polyploidizing mitoses, i.e. mitoses resulting in polyploidy, were found in JTCs (Figure 5). Thus, there occured either acytokinetic mitoses after chromosome segregation in ana-telophase (Figure 5C,D). Binucleate cells generated in that way (Figure 5E,J) can, in turn, enter mitosis (Figure 5F) and give rise to polyploid cells. So-called restitutional mitoses (Nagl, 1978, 1981) were also encountered. Cytophotometry of the DNA content in mitotic figures (Figure 5 J,K) revealed them to contain both the diploid and polyploid DNA amount: 4c, 8c, and 16c (Figure 6). As seen from Fig. 6, among the mitotic figures of different ploidy, there were all mitotic stages present, from prophase to telophase. Hence, mitosis can proceed completely up to the octaploid level. However, Figure 7 demonstrates a predominance of the earlier stages (pro- and metaphases) over the later ones (ana- and telophases) among the polyploid cells. This suggests that mitosis does not proceed up to the end in a part of polyploid cells but it may be blocked at meta- or anaphase. Such a blocked or uncompleted (or restitutional) mitosis may lead to further polyploidization (Nagl, 1978, 1981, Brodsky and Uryvaeva, 1985). As the cell can enter mitosis up to 16c (8n), there are reasons to believe that the 16-32c ploidy level is achieved via endoreduplocation (Figure 4).
From DNA cytophotometry, it follows that the labyrinth trophoblast cells (LTCs) had the ploidy levels 4-32c (Figure 8) similar to those of JTCs Mitotic figures also corresponded to 4c, 8c, and 16c (Figure 9), which indicates mitotic activity up to the octaploid level. Thus, regularities of genome multiplication in the LTCs appear to be similar to those in the JTCs.
Taken together, the data of cytophotometric determination of ploidy of mitotic figures and their morphological features as well as 3H-thymidine uptake and nuclear ploidy levels indicate that in the cambial parts of placenta the trophoblast cells undergo primary polyploidization up to octaploid level via incompleted polyploidizing (restitutional) mitoses and reach higher ploidy degrees (up to 32c) by means of their passing to the endoreduplication cycle. As mitoses in JTCs cease at 15 dpc, the progressive genome multiplication from 16 to 18 dpc appears to occur via endoreduplication (Figure 4).
Discussion Phagocytic activity of trophoblast cells plays a key role in implantation and placenta formation and appears to be one of protective mechanisms against immune attack of maternal allogenic organism (Stewart, Mukhtar, 1988, Lala et al., 1988, Lin et al, 1993).pGTCs and sGTCs phagocytose and utilize intracellularly the uterine epithelium, components of endometrial stroma, and blood cells. Thereby they accomplish: 1) trophic function that is the most evident in the first half of pregnancy; 2) “sanitary” function owing to phagocytosis of the remnants of disintegrated cells by the trophoblast cells to clear thereby the implantational cavity from debris; 3) protective function. The trophoblast invades aggressively and hence injuries the endometrial stroma. A part of trophoblast cells die soon, however, a majority of giant trophoblast cells even attacked by leukocytes remain viable, whereas leukocytes are phagocytosed by giant trophoblast cells (Maximov, 1898, Zybina, 1986). In this connection, some authors suggest a similarity of the decidual reaction with inflammation as a response to penetration of the embryo as a foreign object (Maximov, 1898, Fausek, 1913, Zybina, 1986, Welsh and Enders, 1985, Cross et al., 1994).
The trophoblast cell capability for lysis is connected with expression of different proteases, among them metalloproteinases (MMP) have been studied in detail (for review, see Bishof and Campana, 1997, Kaufmann and Castilucci, 1997). Invasion is facilitated by metalloproteinases that take part in lysis and degradation of decidualizing endometrium; it is balanced by expression of tissue inhibitors of metalloproteinases (TIMP) in decidua (Babiartz et al., 1992, 1996, Leco et al, 1996, Sharkey et al., 1996). A stage-specificity of the proteinase expression has been shown. Thus, MMP-9 transcripts have been found in murine giant trophoblast cells beginning from the 6 dpc. By 8.5 dpc the MMP-9 expression was restricted to giant cells adjacent to the maternal side of the developing placenta, and by the day 9,5 few MMP-9 positive cells remained. Similar regularities were found in the expression of TIMP-3. Thus, transcripts encoding TIMP-3 were detected from day 6-7 in the maternal decidua immediately adjacent to embryonic cells; the degree of TIMP-3 decreased during the sybsequent days parallel to the MMP-9 expression by trophoblast cells (Leco et al, 1996). Metalloproteinase activity in the early embryo is modulated by several growth factors (EGF) and cytokines that take part in the establishment of the correct temporal program of proteinase expression (Harvey et al., 1995). Existence of such program seems to be dictated by the necessity of blastocyst penetration into the endometrium at the early developmental stages.
The results of the present study indicate unambiguously a definite «time schedule” of expression of invasive and phagocytic activities of trophoblast cells as well as of quantitative and qualitative phagocytosis characteristics that strongly correspond to the developmental stages. Thus, pGTCs are able to lyse and phagocytose the endometrial cells at the beginning of implantation (from 7 to 10 dpc), i.e. before the start of their degeneration. The sGTC invasive and phagocytic activities last much longer, from 7 to 18 dpc; different forms of invasion and phagocytosis change each other during this period, first by a gradual increase and then by a decrease. Within this period, lysis of the endometrial epithelium is replaced by degradation and phagocytosis of decidual and blood cells. Intracellular phagocytosis occurs from 7 to 14 dpc, which also was earlier reported by Jollie (1981); it is gradually replaced by the «group phagocytosis» (up to 18 dpc).
The revealed spatiotemporal specificity of invasive and phagocytic activity proved to strongly correspond to the giant trophoblast cell capability for cell reproduction and its mechanisms. Thus, the pGTCs and sGTCs that are in the closest contact with allogenic endometrial cells (epithelium, decidual cells) lose their capability for mitotic division and pass to the endoreproduction (Zybina, 1986, Zybina and Zybina, 1996). The cell cycle is reduced to two stages, S and G, while mitosis is lost completely. Passing through a series of endoreduplication cycles that lead to genome multiplication up to 512-1024c coincides in time with the most marked invasion and active phagocytosis (7-10 dpc in pGTCs and 10-14 dpc in sGTCs).
At present, it is known that the low-differentiated, non-invasive trophoblast cells, as a rule, are able to proliferate, while in the course of differentiation, they progressively lose their proliferative phenotype and acquire invasive phenotype. This regularity has been demonstrated most definitely in placenta of human and other primates by using different markers of proliferation, i.e. 3H-thymidine, proliferative cell nuclear antigen (PCNA), Ki-67, etc. (Bulmer et al., 1988, Muhlhauser et al., 1993, King and Blankenship, 1993, Blankenship and King, 1994).
The invasive phenotype is characterized by expression of a definite
series of integrins (for example,
a 5ß 1, a 1 ß 1), extracellular matrix
components, metalloproteinases. In contrast, the proliferative phenotype has a different
integrin repertoire ( a6 ß4)
as well as connexins and cadherins; expression of different metalloproteinases differs in
the proliferative and invasive trophoblast cells (for review, see Bischof and Campana,
1997, Kaufmann and Castelucci, 1997). In the murine trophoblast cells both in vivo and
in vitro, expression of adhesive molecules and metalloproteinases characteristic of
invasive phenotype has also been found (Damsky et al., 1993, Sutherland et al.,
1993, Babiartz et al., 1996, Leco et al., 1996, Sharkey et al., 1996,
and others).
Trophoblast invasion in murine blastocyst culture has been reported to be accompanied by transformation into the giant cells (i.e. invasive phenotype) and by proliferation of cells of the ectoplacental cone (i.e. proliferative phenotype), a part of them being subsequently transformed into the secondary giant trophoblast cells (Suenaga et al., 1996). Expression of MT1-MMP closely connected with invasion has been demonstrated in the giant trophoblast cells in the cultured murine blastocyst and ectoplacental cone (EC), whereas compact inner cells of EC were MT1-MMP-negative (Tanaka et al., 1998).
Invasive and proliferative phenotypes are stimulated by different growth factors. Thus, in human placenta, CSF-1 and VEGF stimulate proliferation of extravillous trophoblast, while invasion is not significantly affected (Lala, 1997).
The balance between proliferation and differentiation into nonproliferative giant cells, as J.Cross with co-authors notify in their review (Cross et al, 1994), is regulated by trophoblast-specific transcription factors of the helix-loop-helix (HLH) family. Thus, the Mash-2 gene is an important regulator of trophoblast proliferation, and its mutation results in an increase in the number of giant cells at the expence of the proliferative cell population of spongiotrophoblast (Guillemot et al, 1994). Overexpression of another gene, Hxt, in the rat trophoblast stem cell reduces their proliferation and promotes differentiation (Cross et al, 1994).
Meanwhile, up to now no possibility of dedifferentiation of the giant trophoblast cells into the proliferative trophoblast cells have been demonstrated.
In the rat placenta, the cambial trophoblast cell populations, JTCs and LTCs, i.e. derivatives of EC, have been characterized in the current work. They are capable for both DNA synthesis and mitosis and, hence, represent proliferative phenotype. The giant trophoblast cells that can be considered invasive phenotype do not divide mitotically, while continuing chromosome reproduction up to final stages of differentiation, when invasive activity is lost. In this case, endoreduplication, most likely, allows the fast growth of the giant trophoblast cell population to be combined with the invasive activity of these cells.
What is significance of such a way of the trophoblast cell genome multiplication? As trophoblast and endometrial cell closely contact each other (Enders and Schlafke, 1969, Tachi et al., 1970, Tachi and Tachi, 1979, Smith, Wilson, 1974), their mutual injuries may affect chromosome apparatus. We believe it reasonable to put forward a hypothesis that the repeated genome duplication protects the cells from consequences of changes in their hereditary apparatus. It is known that mutation frequency can rise with the introduction of foreign DNA into the organism (Guershenson et al., 1975). Preservation of the nuclear envelope during the whole cell cycle prevents contact of the trophoblast cell genome with DNA of phagocyted cells to thereby decrease probability of mutations.
It is essential to note that sGTCs reach the highest ploidy levels, 128-1024c, at a period of decline of their invasion, when they become fairly stationary and form an almost continuous layer on the border with decidua basalis. On reaching their giant size, the sGTCs seem to stop their migration through the endometrial stroma, so that their slight migration can occur only via lacunae.
Comparison of ways of reproduction of the four trophoblast cell populations studied has shown a reverse relationship between the capability for mitoses and the invasive and phagocytic activities. The JTCs and LTCs that are non-invasive and isolated from endometrium are able to divide mitotically for a long time. However, they still have some contact with allogenic maternal blood cells and tend, although to a lesser degree, to become polyploid: first via incompleted polyploidizing mitoses and then by passing to endoreduplication at a period of final differentiation. This is especially true for the trophospongium cells that face maternal blood and seem to reach the higher ploidy levels than glycogen ones. Therefore, the protective role of polyploidy is likely to be of a different extent and to depend on their functioning to establish contact with allogenic maternal organism. It cannot be ruled out that the way of cell reproduction is determined by their relationship with surrounding tissues. Thus, the trophoblast cells of invasive phenotype, which are apposed to decidua, undergo endoreduplication and become highly polyploid (up to 1024c), while the cells of proliferative phenotype, which contact each other or predominantly anuclear maternal blood cells, can divide mitotically for a long time to reach the lower ploidy level 16-32c.
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Mitoses in junctional zone of rat placenta at 13 dpc. A - diploid metaphase, B - telophase of the diploid cell, C,D - lack of cytokinesis ; E, left - binucleate cell; E,F - union of the two chromosome sets in prophase; G - polyploid metaphase, H - polyploid telophase, I - binucleate cells with polyploid nuclei; J - diploid metaphase, K - octaploid metaphase. A - I - Heidenhein hematoxylin, J, K - Feulgen staining, DNA content has been measured cytophotometricaly.
Percentage of diploid (shaded columns) and polyploid (cross-hatched columns) mitotic figure correspondidng to early (pro- and metaphase, p,m) and late (ana- and telophase, a,t) stages of mitosis in JTCs. A - 13 dpc, B - 14 dpc. Ordinate: percentage of cells.
