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EARLY PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG

April 2000
Volume IV, Number 2
ISSN: 1537-6583
Pages: 099-109

Zybina T. G. and Zybina E. V., Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia

Short title: tGTC genome multiplication

Key words: trophoblast, proliferation, polyploidization, invasion, placenta, vasculature,rodents

Correspondence: Dr. T.G.Zybina, Laboratory of Cell Pathology, Institute of Cytology RAS, 4 Tikhoretsky ave., St.Petersburg 194064, Tel.7(812) 248-1859,
Fax: 7(812) 247-0341, e-mail: zybina@mail.cytspb.rssi.ru

Acknowledgments: This work was supported by the Russian Foundation for Basic Research (grant N 98-04-48322). Translation and editing of the manuscript by Dr. L.Z. Pevzner is gratefully acknowledged.

Abstract

Processes of invasion of the tertiary giant trophoblast cells (tGTCs) into the uterine wall in the course of implantation of the rat embryo at 10-15 days post coitum (dpc) were studied. Reproduction of tGTCs was evaluated using ³H-thymidine incorporation and cytophotometric measurement of the nuclear DNA content. 1.Patterns of trophoblast invasion. Two patterns of invasion were observed: endovascular (from 10 to 15 dpc) and interstitial (from 12 to 15 dpc), via extracellular matrix of endometrial stroma towards the central arterial channel. The both types of the tGTC invasion seem to take part in modification of the arterial wall. The tGTCs were shown to differentiate from cambial trophoblast cells of ectoplacental cone (EC) or junctional zone of placenta. 2. Peculiarities of reproduction of tGTCs. By the start of the endovascular and interstitial migration, tGTCs lose completely their capability for mitotic divisions and are found autoradiographically to stop incorporation of ³H-thymidine soon after beginning of their migration. Cytophotometric measurements of the nuclear DNA content have shown tGTCs to be polyploid. Their degree of ploidy is mainly 8-32c, which is by 1-2 classes of ploidy higher than that of the initial cambial cell populations of ectoplacental cone (EC) and junctional zone trophoblast cells (JTCs). Since the replication processes in tGTCs are limited, their polyploidization seems to occur within cambial populations of the trophoblast cells. The final genome multiplication cycles in the beginning of the tGTC invasion can be accomplished only via endoreduplication, i.e., with the complete elimination of the mitotic mechanism. The not too high level of tGTC ploidy, 8-32 c, probably does not prevent the tGTC deep invasion against the blood flow and through the extracellular matrix and may be of a protective significance in their interrelations with allogenic maternal tissues.

Introduction

Trophoblast invasion and uterine vasculature modification are important characteristics of mammalian placentation, which have been studied in a number of animals and human (Orsini, 1954; Pijnenborg, 1974; Ramsey and Donner, 1908; Pijnenborg et al., 1981; Blankenship et al., 1993, 1996; Maquoi et al., 1997).

Endometrial arteries are invaded by trophoblast, and thereby the definitive placental blood supply is established. However, little is known so far about the relationship between the trophoblast cell capability for a deep migration into endometrium and the proliferative activity as well as other ways of cell growth and reproduction. In human placenta, the deeply invading, intravascular trophoblast cells never incorporate ³H-thymidine, nor are they immunostained with antibodies to the proliferative markers Ki-67/MIB-1 (Kaufmann and Castelucci, 1997).

In the developing rodent placenta, it is the tertiary giant trophoblast cells (tGTCs) that invade deeply into arteries and the decidualized stroma of the endometrium (Orsini, 1954).

The goal of the present work was to find out possibilities of reproduction of tGTCs at the period of their invasion into decidua basalis in the course of placentation in rats as well as to examine in detail the tGTC migration from the ectoplacental cone (EC) and subsequently from the heterogeneous population of trophoblast cells of the junctional zone of placenta. Since tGTCs never divide mitotically, it was of importance to elucidate possibilities and mechanisms of their reproduction during their invasion into decidua basalis.

Materials and Methods

Morphological examination of tGTCs in placenta of randombred white rats was combined with autoradiographic and cytophotometric studies of these cells.

Light microscopy
Pieces of the rat placenta at 14 and 16 day post coitum (dpc) were fixed with a mixture of ethanol¾ glacial acetic acid (3:1), dehydrated in ethanols, and embedded in paraffin. Sections were stained with methyl green¾ pyronin and with Boemer hematoxylin with eosin. A part of preparations were treated with PAS reaction for detection of glycogen.

Autoradiography
Pregnant female rats at the 14th and 15th days of pregnancy were injected with ³H-thymidine (0.5-1 m Ci/g body weight). Two rats were taken for each pregnancy stage. After 2 hr, the placental material was fixed with the 3:1 ethanol¾ glacial acetic acid mixture, dehydrated in ethanols, and embedded in paraffin. Sections, 5 m m thick, were covered with an emulsion of the P type (NIKFI, Moscow, Russia). The sections were exposed for 20 days at 4° C. After development and fixation, preparations were stained with Mayer hematoxylin. In the preparations, index of labeled nuclei (500 trophoblast cells at each stage of development in junctional zone trophoblast cells (JTCs) and 300 cells in tGTCs) was determined.

DNA cytophotometry
Pieces of rat placenta at 12, 13, and 14 days of pregnancy were fixed with the 3:1 ethanol¾ glacial acetic acid mixture and dehydrated in 96% ethanol. in. From them, permanent squash preparations were prepared. For this purpose, the placenta pieces were macerated in the 45% acetic acid, placed on the object glass, covered with coverslips and placed on the dry ice (CO2). The coverslips were removed from the frozen preparations, and these were dried and Feulgen-stained (hydrolysis in 5 N HCl for 30 min at room temperature). As a standard of the haploid and diploid DNA contents, Feulgen-stained smears of rat spermatozoa and peripheral blood lymphocytes were used. Cytophotometric determinations of the DNA content in nuclei of the EC and tGTCs were performed using a Morphoquant (Germany) automated image analyzer composed of a scanning microscope and computer. The total of 1400 nuclei were analyzed. Results of the measurements were processed statistically.

Results

In the rat embryo, the tertiary giant trophoblast cells (tGTCs) are first revealed at 10 dpc. They arise from the EC apical part and migrate on the inner surface of the central lacuna. The trophoblast cells, in the course of their differentiation into tGTCs, are separated from the EC, become rounded and clearly outlined, with the strongly basophilic cytoplasm and rounded nuclei (Figure 1). This migration of tGTCs in lacuna can be asymmetrical: on one side it is faster than on the other; in the latter, the endothelium free of attached tGTCs can be seen. This migration of tGTC is restricted initially by the central lacuna (Figure 1a) and later, by the central arterial channel (Figure 1c); along this channel, they migrate from the junctional zone of placenta.

During subsequent days of development, migration of tGTC is traced along the central arterial channel from the fetal part of placenta (Figures 1c, 2) to the zone of myometrium (Figure 1b), i.e., against the arterial blood flow. Most endovascular tGTCs are revealed in the vessels within the decidualized endometrium and at the endometrium¾ myometrium interface, however, they can also be seen in the myometrial part of the artery. Beginning from 13 dpc, tGTCs shifted endothelium of these vessels, as reported earlier by Enders and Welsh (1993). Whereas within the fetal part of placenta, tGTCs can form a multilayer sheath of the artery (Figure 1c), in the maternal part of placenta they form most often one layer attached to the artery inner wall (Figure 1b,c). Nevertheless, in some cases, clumps of tGTCs that partially fill the arterial lumen can be seen (Figures 1b, 3c); however, they do not seem to affect the blood flow.

Although tGTCs contact with blood, we failed to observe phagocytosis of blood cells by endovascular tGTCs. It should be noted that in the fetal part of the placenta, beneath the arterial sheath of tGTCs, there is a layer of trophoblast glycogen cells that migrate from the placenta junctional zone (Figure 2). In the placenta maternal part, this glycogen cell layer is absent, while adjacent to the arteries is a «muff» of granulated metrial gland cells (GMGCs) (Figures 1b, 2). Occasional GMGCs can migrate into the arterial lumen. The PAS-reaction demonstrated tGTCs to be glycogen-free, whereas the underlying GMGCs might contain glycogen granules. Thus, in the zone of contact of tGTC with decidual tissue, the layers of glycogen-containing cells are located (the glycogen cells in the fetal, while GMGCs, in the maternal part of placenta; Figure 2).

Migration of tGTCs in the rat placenta can be not only endovascular but also interstitial. At 14 and 15 dpc, a part of tGTCs migrate from the junctional zone of placenta through the layer of secondary GTCs into the endometrial stroma towards the central arterial channel (Figures 1c, 2, 3d). A part of tGTCs degenerate, while some of them are concentrated around the arterial periphery. It cannot be ruled out that tGTCs can invade the arterial wall from outside and thereby penetrate into the arterial lumen.

It should be noted that the invading tGTCs never divide mitotically. The autoradiographic study has shown that at the period of their active migration (14 dpc), tGTCs can incorporate the ³H-thymidine label only at the moment of their separation from the placenta junctional zone (Figure 3a). Comparison of Figures 3a and 3b shows that whereas in the JTCs the majority of cell nuclei (70%) include ³H-thymidine, in the zone of the tGTC separation much fewer labeled nuclei (3%) were revealed. In the course of their migration into decidua basalis, tGTCs rapidly lose capability for the DNA synthesis, so they are not labeled with ³H-thymidine almost along the entire central arterial channel (Figure 3c).

The large size of the tGTC nuclei indicates them to be polyploid. To determine the tGTC ploidy level, the nuclear DNA content was measured cytophotometrically at 12-14 dpc. The ploidy level has turned out to progressively rise in the course of development of placenta. At 12 dpc, most tGTC nuclei corresponded to 4-8c, while at 13-14 dpc, 8-16c, with the appearance of a significant percentage of 32c nuclei. As compared to actively proliferating, initial cambial JTC populations, in which polyploidization also occurs, tGTCs have ploidy that is higher, on average, by 1-2 classes (Figure 4). Since, according to autoradiographic data, tGTCs lose capability for DNA replication, their genome multiplication hardly might take place in the course of their migration. This suggests that it is more polyploid trophoblast cells from the placenta junctional zone, incapable of dividing mitototically, which can differentiate into the highly invasive tGTCs migrating inside endometrium.

Disscussion

Placentation in rodents, like in other mammals with haemochorial placenta, is characterized by an extensive invasion of trophoblast cells into endometrium. It is tGTCs that have the deepest invasion. Invasion of the arterial wall brings about substantial physiological changes that are important for the appropriate blood supply of the developing placenta (Blankenship et al., 1993, 1996; Pijnenborg et al., 1996a,b, Meekins et al., 1997). The absence of modification of the arterial wall can lead to complications of the pregnancy, such as hypertension, retarded embryo growth (Blankenship et al., 1993), preeclampsia (Pijnenborg et al., 1996a,b), etc.

The data of this work indicate two types of the rat tGTC invasion: 1) the major one is endovascular i.e. along the inner surface of the arterial wall against the blood flow, and 2) interstitial, via the extracellular space of decidua basalis towards the central arterial channel. In the human and macaque placentas, the both types have been revealed (Pijnenborg et al., 1981, Blankenship et al., 1993a,b, 1996), while in several rodent species studied (mouse, rat, hamster), predominant is the endovascular type of invasion (Orsini, 1954, Pijnenborg et al., 1974, 1981).

Denker (1993) indicates that one of the biological paradoxes of implantation is that the blastocyst trophoblast, when becoming invasive, loses a part of its typical epithelial organization. Thus, its apical plasma membrane acquires adhesiveness to the uterine epithelial cells, which is not characteristic of the epithelial type of the cell organization. Nevertheless, the trophoblast cells do not lose their contacts on the lateral membrane, so they can migrate as sheets rather than as individual cells. We believe this way of invasion to occur in the primary and secondary giant cells of the rat trophoblast. It cannot be ruled out that this pattern of invasion is also characteristic of tGTCs, although a part of them are separated from the total stratum and migrate as individual cells across the endometrial stroma. In the latter case, tGTCs seem to express a more evident mesenchymal phenotype.

The deep invasion of endometrium by the trophoblast cannot be ruled out to play a certain role in prevention of the reaction of rejection of the potentially antigenic embryo by the maternal organism. Probably, the invading trophoblast cells produce hormones that control the immune response of endometrium (Pijnenborg et al., 1974).

The complex character of the trophoblast¾ decidua relationship most likely requires a definite spatio-temporal arrangement of the trophoblast capabilities for adhesion on the vascular wall surface and for cell migration. In this, an important role is played by expression of adhesive molecules by the trophoblast (Blankenship et al., 1993, 1996; Kaufmann and Castelucci, 1997; Maquoi et al., 1997). The adhesive capability is essential for prevention of the trophoblast cell desquamation by the counterflowing blood stream and of their dissemination by the blood to ectopic sites of the maternal organism.

Of similar significance, in our opinion, is also the restriction of the trophoblast cell reproduction. Indeed, the «ban on mitoses» may exclude the possibility of growth and expansion of the trophoblast cell population inside ectopic organs of the maternal organism. As shown in the present work, tGTCs in the rat placenta never divide mitotically; they lose their capability for DNA replication almost from the very beginning of migration into endometrium, which rules out possibility of their migration inside maternal tissues. In human placenta, the deeply invasive intravascular trophoblast is known to never incorporate ³H-thymidine, nor is it labeled with proliferation markers (Blankenship and King, 1994)

The results of the current study indicate a higher degree of the tGTC polyploidy as compared with that of the initial cambial JTC population. Cessation of the DNA replication in tGTC indicates that it is JTCs with a sufficiently high ploidy which differentiate into tGTCs. As shown earlier (Zybina et al, 1996), JTCs undergo polyploidization via incomplete polyploidizing mitoses until the 8c level; after that, the further genome multiplication (the late polyploidization stages) occurs only by endoreduplication, i.e., with the complete absence of mitoses. This cannot be ruled out to minimize the possibility of the spontaneous trophoblast cell proliferation inside maternal tissues.

The trophoblast cell polyploidization in the rapidly developing rodent placenta allows the cells to combine processes of growth and invasion in the absence of proliferation. At the same time, the polyploidization may play a protective role in contact of allogenic tissues (Zybina and Zybina, 1996, Zybina et al, 1996). Since the contact with maternal cells may lead to a damage of tGTCs, the multiple genome doubling in this case seems to serve an additional garantee of preservation of the cell viability.

On the other hand, migration of too large, highly polyploid cells might have occluded blood vessels. Therefore, the ploidy level of 8-32c seems to be optimal for differentiation of tGTCs, allowing them to perform their specific functions.

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References

Blankenship, T. N., Enders, A. C., King, B. F. (1993). Trophoblastic invasion and the development of uteroplacental arteries in the macaque: immunohistochemical localization of cytokeratins, desmin, type IV collagen, laminin and fibronectin. Cell Tiss. Res., 272, 227 - 236

Blankenship, T. N., King, D. E. (1994). Developmental expression of Ki-67 antigen and proliferating cell nuclear antigen in macaque placenta. Develop. Dynam., 201, 324 - 333

Blankenship, T. N., King, D. E. (1996). Macaque intra-arterial trophoblast and extravillous trophoblast of the cell columns and cytotrophoblastic shell express neural cell adhesion molecule (NCAM). Anat. Rec., 245, 525 - 531

Denker H.-W. (1993). Implantation: a cell biological paradox. J.Exper. Zool., 266, 541-558

Enders, A. C., Welsh, A. O. (1993). Structural interaction of trophoblast and uterus during hemochorial placenta formation. J. Exper. Zool., 266, 578 - 587

Kaufmann, P., Castelucci, M. (1997). Extravillous trophoblast in the human placenta. Trophoblast Research, 10, 21 - 65

Maquoi, E., van den Brule, F.A., Castronovo, V., Foidart, J.-M. (1997). Changes in the distribution pattern of galectin-1 and galectin-3 in human placenta correlates with the differentiation pathways of trophoblasts. Placenta, 18, 433-439

Meekins, J. W., Luckas, M. J., Pijnenborg, R., McFadyen, I. R. (1997). Histological study of decidual spiral arteries and the presence of maternal erythrocytes in the intervillous space during the first trimester of normal human pregnancy. Placenta, 18, 459 - 464

Orsini, M. (1954). The trophoblastic giant cells and endovascular cells associated with pregnancy in the hamster, Cricetus auratus. Amer. J. Anat., 94, 273 - 321

Pijnenborg, R., Robertson, W. B., Brosens, I. (1974). The arterial migration of trophoblast in the uterus of golden hamster, Mesocricetus auratus. Reprod. Fert., 40, 269 - 280

Pijnenborg, R., Robertson, W. B., Brosens, I., Dixson, G. (1981). Review article: trophoblast invasion and the establishment of haemochorial placentation in man and laboratory animals. Placenta, 2, 71 - 91

Pijnenborg, R., Luyten, C., Vercruysse, L., Van Assche, F. A. (1996a). Attachment and differentiation in vitro of trophoblast from normal and preeclamptic human placenta. Am. J. Obstet. Gynecol., 175, 30 - 36

Pijnenborg, R., D’Hooghe, T., Vercruysse, L., Bambra, C. (1996b). Evaluation of trophoblast invasion in placental bed biopsies of the baboon, with immunohistochemical localisation of cytokeratin, fibronectin and laminin. J. Med. Primatol., 25, 272 - 281

Zybina, T. G., Zybina, E. V., Stein, G. I., Severova, E. L., Dyban, A. P. (1996). Dynamics of polyploidization and quantitative studies of Ag-NORs in the interphase nucleoli of cambial trophoblast cells in the developing rat placenta. Caryologia, 49, 137 - 152

Zybina, E. V., Zybina, T. G. (1996). Polytene chromosomes in mammalian cells. Intern. Rev. Cytol., 165, 53 - 119

Figure 1

Invasion of tGTCs into decidua basalis at 10 (a) and 15(b,c) dpc in the rat placenta. a - tGTCs (double arrowhead) migrate along the inner surface of the implantational camera and central lacuna deep into decidua basalis. b,c- tGTCs migrate along the inner surface of the arterial wall and reach myometrium. EC - ectoplacental cone, S - secondary giant trophoblast cells, E - embryo, arrowhead - GMGCs. a – methyl green-pyronin; b,c - Boemer hematoxylin staining.

Figure 2

Scheme of rat placenta with particular attention to tGTC location. DC - decidual cells, M – myometrium, GMGCs - granulated metrial gland cells, MS - maternal sinuses, CAC - central arterial channel, tGTCs - tertiary giant trophoblast cells, TSP - cells of trophospongium surround maternal blood lacunae, G - glycogen cells.

Figure 3

Patterns of DNA replication and migration of JTCs and tGTCs in rat placenta at 14 dpc. A - only a few tGTCs (double arrowhead) are ³H-thymidine-labelled in a site of separation from the junctional zone of placenta; B - most of JTC nuclei are labelled; C - endovascular tGTCs (double arrowhead) observed at the level of boundary between endometrium and myometrium show no H-thymidine labelling; D - invasion of tGTCs (double arrowheads) through the extracellular matrix of the decidualized endometrium. a–c - Mayer hematoxylin, d – methyl green–pyronin staining

Figure 4

Percentage of tGTC nuclei of different ploidy level in the rat placenta at 12(a), 13(b) and 14(c) dpc in comparison with initial cell populations. 1 - tGTCs, 2 - initial proliferative cell population of ectoplacental cone (a) and JTCs (b,c), 3 - ectoplacental cone as a whole. Abscissa: ploidy level, c. Ordinate: percentage of nuclei.