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Keywords:

  • glycogen;
  • foeto–maternal interface;
  • invasion;
  • polyploidisation;
  • proliferation;
  • trophoblast

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements and funding
  7. Conflicts of interest
  8. References

In the field vole Microtus rossiaemeridionalis, like in other rodents, invasive secondary giant trophoblast cells (SGTC) form a continuous layer at the foeto–maternal interface in the beginning of placentation. However, in the field vole, at midgestation, clusters of junctional zone (JZ) trophoblast non-giant cells interrupt SGTC layer and progressively replace SGTC at the border of decidua basalis. As a result, ‘border’ cells form a continuous stratum of cytokeratin-positive glycogen-rich cells at the foeto–maternal interface. SGTC plunge into JZ and line the lacunae with maternal blood. SGTC are bound by their highly cytokeratin-positive sprouts forming a framework that holds other trophoblast cell populations. According to DNA cytophotometry, the ‘border’ cells show the highest ploidy among the JZ cells (up to 46% of 8c cells). Thus, in M. rossiaemeridionalis the role of barrier between semiallogenic foetal and maternal tissues is shifted from the highly endopolyploid (32c-1024c) SGTC to the specific subpopulation of glycogen-rich non-giant (2c-16c) ‘border’ trophoblast cells that, however, exceed the ploidy of the deeply located and/or proliferative JZ trophoblast cells.


Abbreviations
SGTC

secondary giant trophoblast cells

JZ

junctional zone of placenta

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements and funding
  7. Conflicts of interest
  8. References

Much attention is being paid to invasion pathways in rodent placenta that serves a model for human placentation. In rodents, the trophoblast invasion includes several steps and involves a range of subtypes of trophoblast cells (Zybina et al., 2000; Adamson et al., 2002; Ain et al., 2003; Arroyo et al., 2005; Vercruysse et al., 2006) that differ in invasion mode and depth, as well as invading cell organisation. Thus, the continuous layer of highly endopolyploid (32c-2048c) secondary giant trophoblast cells (SGTC) capable of phagocytosing components of decidualized endometrium form a physical and physiological barrier between semiallogenic maternal and foetal parts of placenta (Orsini, 1954; Welsh and Enders, 1985; Zybina and Zybina, 2005; Bevilacqua et al., 2010), thereby retaining reproductive capability of genome via endoreduplication (Zybina and Zybina, 2005). The strength of the barrier, most probably, is accounted for by the abundance of intermediate cytokeratin filaments in the peripheral cytoplasm and sprouts of SGTCs in rat placenta (Tamai et al., 2000; Zybina et al., 2011).

Besides, the deeper endovascular and interstitial migration of non-giant trophoblast cell subtypes is seen at later stages of gestation in the placenta of mouse (Adamson et al., 2002) and rat (Zybina and Zybina, 2000, 2005; Ain et al., 2003; Caluwaerts et al., 2005; Vercruysse et al., 2006).

In other mammals, the patterns of foeto–maternal interface in placenta may differ significantly from the mouse and rat. In order to verify universality of the regularities of formation of a barrier to the foeto–maternal interface, we have investigated the modes of trophoblast invasion of another rodent placentation, that is field vole Microtus rossiaemeridionalis.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements and funding
  7. Conflicts of interest
  8. References

Material

Placentas of the East-European field vole M. rossiaemeridionalis bred in a mini-vivarium in the Institute of Cytology RAS at the 9th, 10th, 11th, 13th, 14th and 17th day of gestation were provided by Dr. E. Skholl (five placentas of each stage). The placental junctional zone (JZ) were fixed in ethanol–acetic acid (3:1) and processed either for (immuno)histochemical study or DNA cytometry (see below). All procedures were approved by the Ethics Committee of the Russian Academy of Sciences.

Immunohistochemistry

Iimmunohistochemical testing using the Cytokeratin pan (DAKO, cat. N MO82101) and Ki-67 (DAKO, cat. N IS626) antibodies was carried out according to the standard procedure (Mühlhauser et al., 1993).

Histochemistry

To estimate glycogen localisation, the standard PAS-reaction with diastase control was used (Sheedah and Hrapchak, 1987). To determine cytophotometrically the DNA content, the Feulgen reaction on the squash preparation was carried out as previously described (Zybina et al., 2005).

Microscopy

Slides were examined with an Axiovert 200M microscope with objective lenses 10×/0.30, 20×/0.50, 40×/0.75. The photos were taken with a colour CCD camera Leica DFC 420, format 2592 × 1944. The images were acquired using Photoshop CS2. Mitotic index was estimated as percentage of mitoses in each cell population. Two hundred cells of each subpopulation were analysed from five foetuses at each stage of gestation.

DNA cytometry

DNA content measurement and ploidy estimation was carried out using a ‘Videotest’ image analyser composed of the 83 60 digital CCD-videocamera (Chipper, USA) installed on an EC Bimam-13 microscope and of computer IBM PC 166 as before (Zybina et al., 2005). In each placenta, these were analysed from 150 to 400 trophoblast cell nuclei of each subpopulation and 50 nuclei of foetal erythrocytes as a control of the DNA content per diploid nucleus. Statistical significance of difference in percentage of various ploidy classes between different cell subpopulations at different developmental stages was assessed using the χ2-test (P < 0.01).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements and funding
  7. Conflicts of interest
  8. References

On the ninth day of gestation, the peripheral part of the ectoplacental cone consists of a layer of the SGTC making a continuous ‘front’ of invasion (Figure 1A). Non-giant cells located beneath this layer have a high mitotic activity (mitotic index is 10.1%). By the 10th day of gestation, well-formed JZ and labyrinth could be seen. As early as this stage, clusters of JZ cells begin to interrupt the initially continuous layer of SGTC coming in close contact with decidua basalis.

image

Figure 1. Junctional zone development in placenta of Microtus rossiaemeridionalis. (A) Continuous layer of SGTC is observed at the border with decidua basalis (d) at the ninth day of gestation; the proliferative trophoblast underlies SGTC layer; (B) at the 11th day of gestation accumulations of cytokeratin-positive JZ trophoblast cells (arrowheads) attach decidua basalis (d) thereby replacing SGTC; (C) at the 14th day of gestation SGTC, the ‘border’ cells (bd), spindle-like (sl) and proliferative (pl) trophoblast cell subpopulations of JZ as well as labyrinth (lab) show specific cytokeratin immunolocalisation; decidua basalis (d) is cytokeratin-negative, whereas interstitial invasive trophoblast cells are observed here; (D, E) long cytoplasmic sprouts of SGTC (arrows) surround clusters of the proliferative and spindle-like cells as well as maternal blood lacunae (l), n – nuclei of SGTC. (A) Heidenhein hematoxylin staining; (B–E) Cytokeratin immunostaining with hematoxylin counterstaining of nuclei.

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At the 11–12th day of gestation, progressive replacement of SGTC by the small invasive cytokeratin-positive trophoblast cells migrating from JZ occurs (Figure 1B). The ‘border’ cells differ from the underlying subpopulation by the bulk of cytoplasm in which prominent glycogen deposits can be seen (Figures 2A and 2C). By the 11–12 th day of gestation, the ‘border’ cells completely lost their mitotic activity; in the underlying subpopulation, mitotic activity persisted (mitotic index 4%), after which it progressively attenuated. The ‘border’ cells of JZ facing decidua basalis alternate with SGTC (Figure 1B), the latter being more and more drawn into the depth of the foetal part of placenta.

image

Figure 2. Glycogen-containing trophoblast cells in placenta of M. rossiaemeridionalis at the 12th day of gestation. (A, C, E) PAS-reaction (pink staining) with hematoxylin counterstaining of nuclei (blue); (B) control PAS-staining with diastase digestion. (A) The ‘border’ cells and SGTC show the strongest glycogen staining; it declines in the proliferative subpopulation; labyrinth is mostly glycogen-negative; (C, E) ‘border’ (C) and spindle-like (E) cells differ in glycogen granule deposits (pink); (D, F) ‘border’(D) and spindle-like (F) cells show characteristic cytokeratin arrangement. The designations are the same as at the Figure 1C.

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By the 13–14th days of gestation, the ‘border’ cells form a multilayer stratum at the boundary with decidua basalis (Figure 1C). These cells have large nuclei and abundant cytoplasm, with large intensively stained glycogen deposits (Figure 2C). They show positive cytokeratin immunostaining located mainly at the periphery of cytoplasm, retracing the boundaries of the tightly attached cells (Figures 1C and 2D); and they are Ki-67-negative (data not shown). Under the ‘border’ cell stratum, there is a zone of cells with spindle-like nuclei less rich in cytoplasm, with peripheral and perinuclear cytokeratin localisation (Figures 1C and 1F) and rather small. They are also mitotically inactive and Ki-67-negative. Close to labyrinth, a narrow zone of Ki-67-positive trophoblast cells with a mitotic index 5% is found. The proliferative cells often contain bundles of short cytokeratin filaments that encircle nuclei at one side (Figures 1D and 1E). By the 17th day of gestation, the proliferative subpopulation practically disappears.

In the decidua basalis, some amount of cytokeratin-positive interstitially invading trophoblast cells can be found (Figure 1C).

SGTC (Figures 1B–E) found in the depth of JZ have the most intense cytokeratin immunostaining; it is especially dark at the periphery and in the long sprouts by which SGTC contact each other (Figures 1C–E). The perinuclear staining is also dark. Due to the intense immunostaining of cytokeratin filaments, a kind of system or continuous network of SGTC inserted into the layers of the non-giant trophoblast subpopulations in JZ is seen (Figures 1B–E). Often SGTC, with their long massive sprouts, surround the clusters of the spindle-like and proliferative cells of JZ (Figures 1D and E). They may play a role in the framework that supports and isolates the clusters of functionally different spindle-like and proliferative cells. SGTC, tightly connecting each other, also form a lining of the system of the maternal blood lacunae. In the rat placenta, massive cytokeratin filaments are also found in the SGTC at the border with decidua as well as in the trabecular spongiotrophoblast that line maternal blood lacunae (Zybina et al., 2011). Thus, similarities of cytokeratin arrangement in the functionally similar trophoblast cell populations occur in both rodent species.

Interestingly, trophoblast cell population show similarities in the expression of the PRL/PL gene family, that is prolactins and prolactin-related proteins (Simmons et al., 2008). Thus, expression of Prl5a1 is characteristic of murine trophoblast giant cells in the ectoplacental cone as well as in the well-formed spongiotrophoblast (Simmons et al., 2008). Conceivably, both giant trophoblast subpopulations – murine trophoblast giant cells and spongiotrophoblast – are similar functionally; in particular, both of them form a barrier between semiallogenic foetal and maternal tissues, that is decidua and maternal blood cells.

In the Feulgen-stained squash slides of JZ, three types of cell accumulation are clearly distinguished: (1) the ‘border’ cell with round/ovoid nuclei, (2) the spindle-like cells having, correspondingly, spindle-like nuclei and (3) proliferative cells having small round nuclei lying closely to each other. According to DNA cytometry (Figure 3), at the 11th day of gestation, the proliferative subpopulation contains not only diploid (34.3%), but tetraploid (44.8%) and 8c cells (16.1%), most probably due to polyploid mitoses (Zybina et al., 2005). Non-proliferative ‘border’ subpopulation has in general a higher ploidy level: 2c cells are very rare (1.6%) and the vast majority constitute 4c (53.1%) and 8c (34.7%) cells, probably due to the preferential invasion of cells of higher ploidy. At the 14th day of gestation, ploidy of the proliferative subpopulation does not change significantly (Figure 3). The two mitotically inactive subpopulations of ‘border’ and spindle-like cells reach higher ploidy levels. Thus, the ‘border’ cells are almost devoid of diploid cells (0.8%), whereas 50.4% of them are 4c and 46.3% 8c; there are a few 16c cells (2.5%). In the spindle-like subpopulation the ploidy is a slightly lower compared to the ‘border’ ones. Thus, the share of diploid cells falls to 5.3% whereas 4c cells constituted 57.0%, and 8c 35.8%; there was also a small amount of 16c cells (1.1%) and 32c (1.1%). The difference in percentage of various ploidy classes between different cell subpopulations is statistically significant (χ2-test giving P < 0.01).

image

Figure 3. Dynamics of polyploidisation of different JZ trophoblast cell subpopulations in placenta of the field vole M. rossiaemeridionalis. Noteworthy, in well-developed placenta the majority of the ‘border’ JZ cells are highly polyploid (8c and higher).

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By the 17th day of gestation, when the proliferative subpopulation disappears, the difference in the percentage of cells of different ploidy level between the ‘border’ and spindle-like subpopulations becomes more pronounced due to an increase in highly polyploid cells in the ‘border’ subpopulation (up to 60%; Figure 3). Therefore, a close interrelation between differentiation of a range of JZ trophoblast subpopulations and their polyploidisation appears to be clearly distinctive.

The data show that in the beginning of the field vole placenta formation, like in mouse and rat, a continuous layer of SGTC is formed. The latter separates the foetal part of placenta from the maternal one. However, in the field vole, unlike mouse and rat placentation, JZ trophoblast cells progressively replace initially continuous SGTC layer at the border with decidua as soon as JZ and labyrinth are formed.

In the mouse placenta prior to 12.5 days of gestation, cells that had invaded endometrium beyond the main giant cell layer are tightly located around arteries, but show no general invasion of the interstitium in the decidua and venous sinuses (Adamson et al., 2002). Glycogen trophoblast cells invade the decidua outside the trophoblast giant cell layer after only 12.5 days of gestation, the invasion having a diffuse interstitial invasive pattern (Adamson et al., 2002). Such an invasion of murine trophoblast cells has been confirmed by the expression of MMP9 beginning only from the 13.5 day of gestation (Coan et al., 2006). Taking into account the terms of pregnancy and formation of the main parts of placenta in mouse and field vole are similar, we suggest that invasion of low-ploid JZ trophoblast cells in the field vole starts at the earlier stages of gestation compared to the mouse. It may be accounted for by some peculiarity of trophoblast formation in field vole, for example by the continued proliferation of the trophectoderm (Copp and Clarke, 1988). Another distinction of field vole is that the ‘border’ cells represent a continuous multilayered stratum of cells tightly attached to each other rather than the interstitially invading ones. Nevertheless, interstitial trophoblast invasion of decidua in M. rossiaemeridionalis is also observed; it is clearly distinctive at the 14th day of gestation.

Thus, the role of a barrier at the foeto–maternal interface is shifted to the specific glycogen-rich JZ trophoblast subpopulation of ‘border’ cells. Though the ‘border’ cells do not reach the highest ploidy level characteristic of SGTC (32c-1024c), they exceed the ploidy of the deeply located and/or proliferative cells.

The local arrival of JZ trophoblast cells in close contact to the decidua has also been observed in a range of South American Cricetidae. Thus in Hylaeomys placenta, glycogen trophoblast cells occur at the outer margin of JZ just close to decidua. In Necromys the glycogen cells invades the decidua near the maternal blood channel-entering placenta (Favaron et al., 2011). Further information in this regard will lead to the best understanding of evolution and significance of controlled trophoblast invasion in mammalian placentation.

Polyploidy of trophoblast cells at the border with maternal tissues, as pointed out before (Zybina and Zybina, 2005, 2012), probably plays a protective role in embryo development. Indeed, close contact of semi-allogenic trophoblast and decidual cells may result in mutual damaging, for example to chromosomes. As polyploid trophoblast cells are much more resistant to irradiation (MacAuley et al., 1998), polyploidisation of ‘border’ cells may protect their genome from possible mutagenic effect of breakdown material.

The data presented here confirm the assumption that cytokeratin filaments in the giant trophoblast cells may contribute in the barrier at the foeto–maternal interface in the rat placenta (Zybina et al., 2011). This is confirmed by data that lack of expression of cytokeratin 8 and 19 results in disruption of integrity of the murine giant trophoblast cell layer (Tamai et al., 2000), which result in embryo death. Interestingly, in mutants of Mrj gene involved in cytokeratin assembly, trophoblast cells are characterised by abnormal cell morphology, collapse of the actin cytoskeleton, E-cadherin and β-catenin misexpression and extracellular matrix disorganisation (Watson et al., 2011). Cytokeratin importance in the barrier function of trophoblast is suggested by observation of cytoskeleton arrangement in the human syncytiotrophoblast, particularly in the syncytial nuclear aggregates (SNA). Cytokeratin showed the strongest expression; it is heavily distributed in the SNA, which suggests that SNA are stable structures. Tubulin also is strongly expressed whereas actins are poorly associated with SNA (Coleman et al., 2013).

The diastase-controlled PAS-reaction shows that the ‘border’ cells of JZ of M. rossiaemeridionalis contain prominent glycogen deposits. It suggests that the ‘border’ cells represent a specialised trophoblast subpopulation similar to glycogen cells in the mouse and rat placenta (Zybina et al., 2000; Adamson et al., 2002; Coan et al., 2006; Zybina et al., 2011). However, in mouse and rat, glycogen cells are developed in a form of clusters in the depth of JZ, whereas in M. rossiaemeridionalis glycogen-rich ‘border’ cells are formed at the boundary of the foetal and maternal parts of placenta. As found in the murine placenta, the differentiated glycogen cells express glucagon (Coan et al., 2006), which stimulates breakdown of glycogen to glucose (Lodish et al., 1988). Therefore, it is quite possible that the glycogen-rich ‘border’ cells take part in carbohydrate metabolism and probably provide embryonic tissues by glucose.

The presence of large glycogen deposits in the ‘border’ cells of JZ seems interesting because in human placenta accumulations of glycogen-rich cells are also found within the extravillous trophoblast attached to the decidualized endometrium (Georgiades et al., 2002). On the other hand, human extravillous trophoblast cells also show polyploidisation up to 8c-16 (Zybina et al., 2002). Thus, the data of the present study points out that the regularities of formation of the barrier at the foeto–maternal interface during placentation of field vole may be helpful in understanding the analogous process in human placenta.

Acknowledgements and funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements and funding
  7. Conflicts of interest
  8. References

The authors are grateful to Dr. L.Z. Pevzner for help in translating and editing the manuscript. The study was supported by the Granting Program of the Russian Academy of Sciences for Molecular and Cell Biology.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements and funding
  7. Conflicts of interest
  8. References
  • Adamson SL, Lu Y, Whiteley KJ, Doug H, Hemberger M, Pfarrer C, Cross J (2002) Interactions between trophoblast cells and the maternal and fetal circulation in the mouse placenta. J Devel Biol 250: 35873.
  • Ain R, Canham LN, Soares MJ (2003) Gestation stage-dependent intrauterine trophoblast cell invasion in the rat and mouse: novel endocrine phenotype and regulation. Dev Biol 260: 17690.
  • Arroyo JA, Konno T, Khalili DC, Soares MJ (2005) A simple in vivo approach to investigate invasive trophoblast cells. Int J Dev Biol 49: 97780.
  • Bevilacqua E, Hoshida MS, Amarante-Paffaro A, Albieri-Borges A, Gomes SZ (2010) Trophoblast phagocytic program: roles in different placental systems. Int J Dev Biol 54: 495505.
  • Coan PM, Conroy N, Burton GJ, Ferguson-Smith AC (2006) Origin and characteristics of glycogen cells in the developing murine placenta. Devel Dynam 235: 328094.
  • Coleman SJ, Gerza L, Jones CJP, Sibley CP, Aplin JD, Heazell AEP (2013) Syncytial nuclear aggregates in normal placenta show increased nuclear condensation but apoptosis and cytoskeletal redistribution are uncommon. Placenta 34: 44955.
  • Copp AJ, Clarke JR (1988) Role of the polar trophectoderm in determining the pattern of early post-implantation morphogenesis in mammals: evidence from development of the short-tailed field vole, Microtus agrestis. Placenta 9: 64353.
  • Caluwaerts S, Vercruysse L, Luyten C, Pijnenborg R (2005) Endovascular trophoblast invasion and associated structural changes in uterine spiral arteries of the pregnant rats. Placenta 26: 57484.
  • Favaron FO, Carter AC, Ambrósio CA, Morini AC, Mess AM, de Oliveira MF, Maria A, Miglino MA (2011) Placentation in Sigmodontinae: a rodent taxon native to South America. Reprod Biol Endocrinol 9: 55.
  • Georgiades P, Ferguson-Smith A, Burton GJ (2002) Comparative developmental anatomy of the murine and human placenta. Placenta 23: 319.
  • Lodish H, Baltimore D, Berk A, Zipurlsky SL, Matsudaria P, Darnell J (1988) Molecular cell Biology. New York: Scientific American Books, Inc.
  • MacAuley A, Cross JC, Werb Z (1998) Reprogramming of the cell cycle for endoreduplication in rodent trophoblast cells. Mol Biol Cell 9: 795807.
  • Mühlhauser J, Crescimanno C, Kaufmann P, Hofler H, Zaccheo D, Castellucci M (1993) Differentiation and proliferation patterns in human trophoblast revealed by c-erb-2 oncogene product and EGF-R. J Histochem Cytochem 41: 16573.
  • Orsini M (1954) The trophoblastic giant cells and endovascular cells associated with pregnancy in the hamster, Cricetus auratus. Am J Anat 93: 273331.
  • Sheedah DC, Hrapchak BB (1987) Theory and practice of histotechnology, 2nd edn. Columbus, OH: Bartelle Memorial Institute.
  • Simmons DG, Rawn S, Davies A, Hughes M, Cross J (2008) Spatial and temporal expression of the 23 murine prolactin/placental lactogen-related genes is not associated with their position in the locus. BMC Genomics 9: 35282.
  • Tamai Y, Ishikawa T, Bösl MR, Mori M, Nozaki M, Baribault H, Oshima RG, Taketo MM (2000) Cytokeratins 8 and 19 in the mouse placental development. J Cell Biol 151: 56372.
  • Vercruysse L, Caluwaerts S, Luyten C, Pijnenborg R (2006) Interstitial trophoblast invasion in the decidua and mesometrial triangle during the last third of pregnancy in the rat. Placenta 27: 2333.
  • Watson ED, Hughes M, Simmons DG, Natale DRC, Sutherland AE, Cross JC (2011) Cell-cell adhesion defects in Mrj mutant trophoblast cells are associated with failure to pattern the chorion during early placental development. Dev Dyn 240: 250519.
  • Welsh AO, Enders AC (1985) Light and electron microscopic examination of the nature of decidual cells of the rat, with emphasis on the antimesometrial decidua and its degeneration. Am J Anat 172: 130.
  • Zybina TG, Zybina EV (2000) Genome multiplication in the tertiary giant trophoblast cells in the course of their endovascular and interstitial invasion into the rat placenta decidua basalis. Early Pregnancy (online) 4: 99109.
  • Zybina TG, Zybina EV (2005) Cell reproduction and genome multiplication in the proliferative and invasive trophoblast cell populations of mammalian placenta. Cell Biol Intern 29: 107183.
  • Zybina TG, Zybina EV (2012) Cell cycle modification in trophoblast cell populations in the course of placenta formation. In: Kušik-Tišma E, ed. DNA replication and related processes. Rijeka: InTech, pp. 22758.
  • Zybina EV, Zybina TG, Stein GI (2000) Trophoblast cell invasiveness and capability for the cell and genome reproduction in the rat placenta. Early Pregnancy (online) 4: 3957.
  • Zybina TG, Kaufmann P, Frank H.-G, Freed J, Kadyrov M, Biesterfeld S (2002) Genome multiplication of extravillous trophoblast cells in human placenta in the course of differentiation and invasion into endometrium and myometrium. I. Dynamics of polyploidization. Tsitologiya 44: 105867.
  • Zybina EV, Zybina TG, Bogdanova MS, Stein GI (2005) Whole-genome chromosome distribution during nuclear fragmentation of giant trophoblast cells of Microtus rossiaemeridionalis studies with the use of gonosomal chromatin arrangement. Cell Biol Intern 29: 106670.
  • Zybina TG, Stein GI, Zybina EV (2011) Endopolyploid and proliferating trophoblast cells express different patterns of intracellular cytokeratin and glycogen localization in the rat placenta. Cell Biol Intern 35: 64955.