Polyploidization and endomitosis in giant cells of rabbit trophoblast

Morphological and cytophotometric investigations have been performed on giant cells of the rabbit trophoblast to reveal a mechanism of nuclei polyploidization and define the level of polyploidy. The character of endomitotic chromosomes is found to differ and depend largely on the degree of nuclei polyploidy. Small chromosomes were found in nuclei with low levels of polyploidy. For highly polyploid nuclei, two stages are distinguished. In the first case condensed chromosomes join into bundles resembling Riesenchromosomen in plants, whereas in the second, decondensed chromosomal threads separate and disperse in the karyoplasm. The splitting does not involve nuclei-forming chromosomes in the region of the nucleolar organiser. The degree of polyploidy was determined on the 15th day of development. It was found that giant cell nuclei contain DNA in amounts corresponding to 32-512 chromosomal sets. Most of the nuclei have levels of 128c and 256c. Highly-polyploid nuclei disintegrate into small nuclei with the degree of polyploidy varying from 1c to 32c. Di- tri- and tetraploid nuclei predominate.     

Morphological and cytofluorometric study of the giant cells of the trophoblast of the common vole]

The primary and secondary giant cells of trophoblast in placenta Microtus arvalis were studied. The giant polyploid nuclei are formed in result of series of successively proceeding endomitotic polyploidization of chromosomes. Two stages of endomitosis are described: endointerphase with the uniform net of thin chromatin threads and the stage when small round or rod-shaped paired chromosomes gather mostly under the nuclear membrane. Great number of round, oval, and complex-shaped nucleoli may be seen in nuclei during both stages of endomitosis, the number growing during polyploidization. The morphology of the chromosome-nucleolar apparatus involves peculiarities of the polyploidization mechanism in placenta Microtus arvalis trophoblast. Endomitosis occurs both in low and high-polyploid nuclei. Cytofluorometric determination of the DNA amount in nuclei polyploid nature. The degree of polyploidy of the trophoblast giant cells nuclei during terminal differentiation of placenta corresponds to 128c-512c, and some nuclei contain the DNA amount corresponding to 1024 and 2048 chromosomal sets. The cause of origin of the polyploid cells in trophoblast of rodents placenta is discussed.     

Distribution of chromosome material during division of giant nuclei by fragmentation in rodent trophoblast. Morphologic and cytophotometric study]

A study was made of a population of secondary giant cells (in the placenta of white rats and mice), of which a rather high polyploidy (128c--1024c) is characteristic, and which remains viable up to the end of pregnancy. At a certain stage of cell differentiation, some giant nuclei, looking as interphase nuclei, are divided into numerous smaller nuclear fragments bound with nuclear membranes. Two ways of division have been described: by a progressive budding of small nuclei into the cytoplasm, and the total division of the original nucleus into numerous tightly contracting nuclear fragments. Multinuclear cells originating from the nuclear fragmentation rather soon degenerate. The cytophotometrical measurement of the DNA amount in newly formed fragments has shown their ploidy extending from 1 to 32c, di-, three-, tetra-, and octoploid nuclei predominating. The distribution of chromosomal markers of the interphase nuclei (nucleoli, heterochromatinous blocks of nucleolus-forming chromosomes) confirms the photometrical evidence on the trends of chromosome fragmentation into genes. The fragmentation of the giant nucleus is preceded by a complex rearrangement of genetical material in the original nucleus, resulting in becoming polygenomal from polytene, with individual genomes separating to be segregated again, during division.     

Polytene chromosomes in mouse trophoblast giant cells

Mouse trophoblast giant cells undergo successive rounds of DNA replication resulting in amplification of the genome. It has been difficult to determine whether giant cell chromosomes are polyploid as in liver cells or polytene as in Dipteran salivary glands because the chromosomes do not condense. We have examined the pattern of hybridization of mouse giant cells with a variety of in situ chromosome markers to address this question. Hemizygous markers displayed one hybridization signal per nucleus in both diploid and giant cells, while homozygous markers displayed two signals per nucleus in both cell types. These patterns are consistent with cytological evidence indicating that giant cell chromosomes are polytene rather than polyploid. However, in contrast to the situation in Dipteran salivary glands, the two homologues do not appear to be closely associated. We conclude that the mechanism of giant cell DNA amplification involves multiple rounds of DNA replication in the absence of both karyokinesis and cytokinesis, and that sister chromatids, but not homologous chromosomes, remain closely associated during this process.     

PAL31 expression in rat trophoblast giant cells

PAL31 is a proliferation-related acidic nuclear protein that belongs to the leucine-rich protein family and is expressed cell-cycle-dependently. Trophoblasts differentiate into the trophoblast giant cells (TGCs) through the unusual type of cell cycle, namely endoreduplication. In the present study, we investigated the spatiotemporal pattern of PAL31 expression in rat placenta and Rcho-1 cell line. The PAL31 mRNA concentration varied in different areas of the placenta, and was barely detectable in the TGC layer. In Rcho-1 cells, although the level of PAL31 mRNA decreased dramatically during differentiation, PAL31 was detected even after differentiation. The site of intranuclear localization of PAL31 mostly overlapped with that of PCNA in the undifferentiated Rcho-1 cells, while they were not overlapped in differentiated cells. Thus, the subcellular localization of PAL31 in Rcho-1 cells significantly changed, and loss of cell cycle dependency after differentiation was noted. PAL31 is suggested to play a role in the endoreduplication distinct from the usual DNA duplication.     

Polyploidization dynamics of tertiary trophoblast giant cells in the rat placenta

Polyploidization peculiarities of tertiary giant trophoblast cells during their active detaching from the ectoplacental cone and migrating into decidua basalis are investigated. On the 12th day of gestation, the ploidy of the majority of cell nuclei varies within 4-8c, although there are a few 16c and 32c nuclei. On the 13th and 14th days of gestation, the ploidy level of tertiary giant trophoblast cells enhances; 8c and 16c nuclei prevail, the percentage of 32c nuclei increases, 64c nuclei arising. The ploidy level of tertiary giant cell coincides with the average and/or maximum ploidy degree of precursor cell populations. The significance of polyploidy as indispensable condition of differentiation of the trophoblast cells that actively invade into maternal tissues is discussed.     

Intranuclear and cytoplasmic annulate lamellae in the polyploid giant cells of the trophoblast

Intranuclear and cytoplasmic annulate lamellae in polyploid giant cells of the trophoblast have been studied in rat placenta on days 12--17 of development. The annulate lamellae are present in the cytoplasm within a limited time, being visible on day 12 only. These are arranged in bundles near the nucleus to be moving then to the cytoplasm. The end parts of annulate lamellae are broadened to make cisterns of rough endoplasmic reticulum. Unlike the cytoplasmic annulate lamellae, those found within the nucleus are seen in part of the nuclei investigated throughout the whole period examined to look as single structures (not gathered in bundles), they can be branching, separating closed spaces within the nucleus (making local swellings in the loci of branching; the latter having electron dense or transparent vesicles). Association with nuclear chromatin in some regions is a peculiar feature of the intranuclear annulate lamellae. This association is especially obvious at endoprophase in the cycle ofthe polytene nucleus during the somatic conjugation--chromonemes unite in a bundle and condense. Ultrastructural changes of the annulate lamellae is noted throughout the polytene nucleus cycle and during the cell differentiation. It is supposed that in the case of temporary labile chromosome polyteny in the nuclear cycle, which is characteristic of mammalian trophoblasts, annulate lamellae can well compare, in their function, with the synaptonemal complex--these prevent from too tight associations of homologues in the course of somatic conjugation of chromosomes.     

Functional analysis of trophoblast giant cells in parthenogenetic mouse embryos

Diploid mouse embryos containing only maternal DNA (parthenotes) fail, in part, because the inner cell mass does not induce the trophoblast to grow. In this study, we asked whether any of the defects in parthenotes may arise from alterations in trophoblast function. We examined the expression of genes important for normal trophoblast function and found several trophoblast genes that were expressed at normal levels in the primary trophoblast cells of parthenotes: E-cadherin, a cell adhesion molecule, was expressed normally in both the ICM and trophectoderm of parthenogenetic blastocysts and blastocyst outgrowths; the gene for Hxt, a basic helix-loop-helix factor that regulates trophoblast development, was expressed in both zygotic and parthenogenetic giant cells; placental lactogen-1, a hormone that is normally secreted by trophoblast giant cells, was expressed in most of both parthenogenetic and normal trophoblast cells; and the 92 kDa matrix metalloproteinase, gelatinase B, also known as MMP-9, was secreted at equivalent levels by both zygotic and parthenogenetic blastocyst outgrowths. However, once the outgrowths had developed, a subpopulation of trophoblast cells in parthenogenetic embryos had decreased DNA replication and significantly fewer nucleoli per nucleus than did zygotic embryos. Moreover, the parthenogenetic trophoblast cells growing out from blastocysts had a decreased viability in culture. These data suggest that, although parthenogenetic embryos are able to initiate primary trophoblast differentiation, the stability and continued differentiation of trophoblast giant cells may be abnormal. Our data support the hypothesis that the deficiency of secondary trophoblast giant cells may contribute to the decline of parthenogenetic embryos and suggest that the factors controlling this subset of trophoblast are distinct from those for primary trophoblast. Dev Genet 20:1–10, 1997. © 1997 Wiley-Liss, Inc.     

Diverse subtypes and developmental origins of trophoblast giant cells in the mouse placenta

Trophoblast giant cells (TGCs) are the first terminally differentiated subtype to form in the trophoblast cell lineage in rodents. In addition to mediating implantation, they are the main endocrine cells of the placenta, producing several hormones which regulate the maternal endocrine and immune systems and promote maternal blood flow to the implantation site. Generally considered a homogeneous population, TGCs have been identified by their expression of genes encoding placental lactogen 1 or proliferin. In the present study, we have identified a number of TGC subtypes, based on morphology and molecular criteria and demonstrated a previously underappreciated diversity of TGCs. In addition to TGCs that surround the implantation site and form the interface with the maternal deciduas, we demonstrate at least three other unique TGC subtypes: spiral artery-associated TGCs, maternal blood canal-associated TGCs and a TGC within the sinusoidal spaces of the labyrinth layer of the placenta. All four TGC subtypes could be identified based on the expression patterns of four genes: Pl1, Pl2, Plf (encoded by genes of the prolactin/prolactin-like protein/placental lactogen gene locus), and Ctsq (from a placental-specific cathepsin gene locus). Each of these subtypes was detected in differentiated trophoblast stem cell cultures and can be differentially regulated; treatment with retinoic acid induces Pl1/Plf+ TGCs preferentially. Furthermore, cell lineage tracing studies indicated unique origins for different TGC subtypes, in contrast with previous suggestions that secondary TGCs all arise from Tpbpa+ ectoplacental cone precursors.     

Hypoxia inhibits differentiation of lineage-specific Rcho-1 trophoblast giant cells

Defects in placental development lead to pregnancies at risk for miscarriage and intrauterine growth retardation and are associated with preeclampsia, a leading cause of maternal death and premature birth. In preeclampsia, impaired placental formation has been associated with alterations in a specific trophoblast lineage, the invasive trophoblast cells. In this study, an RT-PCR Trophoblast Gene Expression Profile previously developed by our laboratory was utilized to examine the lineage-specific gene expression of the rat Rcho-1 trophoblast cell line. Our results demonstrated that Rcho-1 cells represent an isolated, trophoblast population committed to the giant cell lineage. RT-PCR analysis revealed that undifferentiated Rcho-1 cells expressed trophoblast stem cell marker, Id2, and trophoblast giant cell markers. On differentiation, Rcho-1 cells downregulated Id2 and upregulated Csh1, a marker of the trophoblast giant cell lineage. Neither undifferentiated nor differentiated Rcho-1 cells expressed spongiotrophoblast marker Tpbpa or labyrinthine markers Esx1 and Tec. Differentiating Rcho-1 cells in hypoxia did not alter the expression of lineage-specific markers; however, hypoxia did inhibit the downregulation of the trophoblast stem cell marker Id2. Differentiation in hypoxia also blocked the induction of CSH1 protein. In addition, hypoxia inhibited stress fiber formation and abolished the induction of palladin, a protein associated with stress fiber formation and focal adhesions. Thus, Rcho-1 cells can be maintained as a proliferative, lineage-specific cell line that is committed to the trophoblast giant cell lineage on differentiation in both normoxic and hypoxic conditions; however, hypoxia does inhibit aspects of trophoblast giant cell differentiation at the molecular, morphological, and functional levels.     

Participation of ezrin in bacterial uptake by trophoblast giant cells

A simple, safe and cost-effective treatment protocol in ovarian stimulation is of great importance in IVF practice, especially in the case of previous unsuccessful attempts. hCG has been used as a substitute of LH because of the degree of homology between the two hormones. The main aim of this prospective randomized study was to determine, for the first time, whether low dose hCG added to rFSH for ovarian stimulation could produce better results compared to the addition of rLH in women entering IVF-ET, especially in those women that had previous IVF failures. An additional aim was to find an indicator that would allow us to follow-up ovarian stimulation and, possibly, modify it in order to achieve a better IVF outcome; and that indicator may be the cDNA copies of the LH/hCG receptor. Group A patients (n = 58) were administered hCG and Group B rLH (n = 56) in addition to rFSH in the first days of ovarian stimulation. The number of follicles and oocytes and, most importantly, implantation and pregnancy rates were shown to be statistically significantly higher in the hCG group. This study has also determined, for the first time to our best knowledge, m-RNA for LH/hCG receptors in the lymphocytes of peripheral blood 40 h before ovum pick-up. cDNA levels of the hCG receptor after ovarian stimulation were significantly higher among women receiving hCG compared to those receiving LH. In addition, higher levels were encountered among women with pregnancy compared to those without, although this was not statistically significant due to the small number of pregnancies. It seems that hCG permits a highly effective and more stable occupancy of rLH/hCG receptors and gives more follicles and more oocytes. The determination of cDNA copies could be, in the future, a marker during ovulation induction protocols and of course a predictor for the outcome of ART in the special subgroup of patients with previous failures.     

Participation of ezrin in bacterial uptake by trophoblast giant cells

Background  

Trophoblast giant (TG) cells are involved in systematic removal of bacterial pathogens from the maternal-fetal interface of the placenta. In particular, TG cells have the ability to take up extracellular antigens by active phagocytosis induced by interferon-gamma (IFN-gamma). We previously reported that heat shock cognate protein 70 (Hsc70) present on the surface of TG cells mediated the uptake of Brucella abortus. However, the mechanism of bacterial uptake by TG cells is not completely understood. Here we identified ezrin, a member of ezrin-radixin-moesin (ERM) protein family, as a molecule associated with Hsc70.     

Localization of placental lactogen-I in trophoblast giant cells of the mouse placenta

The purpose of this investigation was to identify the cellular origin of placental lactogen-I (PL-I) expression in the mouse placenta and to cytologically define the transition from PL-I to PL-II expression during gestation. PL-I mRNA expression was assessed by in situ hybridization, and expression of PL-I and PL-II protein was determined by immunocytochemical analysis. PL-I mRNA and protein were localized to trophoblast giant cells. Trophoblast giant cells ceased producing PL-I at midgestation and began expressing PL-II. PL-I immunoreactivity was present in trophoblast giant cells on Days 9 and 10 of gestation but was not detectable in trophoblast giant cells on Day 11 of gestation. Immunoreactive PL-II-producing giant cells were detected first on Day 10 of gestation, continuing on Day 11 of gestation. Expression of PL-I and PL-II signals a significant functional transition in trophoblast giant cells of the developing mouse placenta.     

Ultrastructural morphology and relaxin immunolocalization in giant trophoblast cells of the golden hamster placenta

Relaxin immunoreactivity was previously demonstrated in three cell types within the hamster placenta; fetal primary and secondary giant trophoblast cells (GTCs) and maternal endometrial granulocytes. The objectives of the present research were to examine the ultrastructure of the GTCs and identify the intracellular relaxin storage site. Primary GTCs, first present on day 8 of gestation, were characterized by numerous polyribosomes and large heterogeneous cytoplasmic inclusions suggesting phagocytic activity. Primary and secondary GTCs from days 10, 14, and 15 of gestation contained numerous polyribosomes, mitochondria with tubular cristae, and extensive Golgi complex, and abundant rough endoplasmic reticulum, all characteristics of a cell actively involved in protein synthesis. Membrane-bound secretory granules were not present. Relaxin was immunolocalized within the Golgi complex of primary and secondary GTCs using the avidin-biotin-peroxidase method. Following differential centrifugation of hamster placental homogenates and radioimmunoassay (RIA) of subcellular fractions, the majority of relaxin immunoactivity was detected in the postmicrosomal fraction; however, the majority of relaxin immunoactivity from similarly treated pig corpora lutea was present in the mitochondrial/granule fraction. These data indicate that hamster placental relaxin is not stored in membrane-bound secretory granules but is contained within the extensive Golgi complex of the GTC.     

Binucleate trophoblast giant cells in the water buffalo (Bubalus bubalis) placenta

The binucleate trophoblast giant cells (BNC) of the water buffalo, Bubalus bubalis, placenta were studied, with emphasis on the synthesis of BNC-specific proteins. Placentomal tissues of 27 water buffalos (2-10 months of pregnancy) were processed for light and electron microscopy. The frequency of BNCs was 20% of the trophoblastic cells in 2-3-month placentas and increased to 27% in the later stages. Ultrastructurally, binucleate cells displayed a prominent granular endoplasmic reticulum and Golgi apparatus, typical of cells involved with protein synthesis and exportation. The buffalo BNCs contained periodic acid-Schiff (PAS)-positive granules and reacted with antisera against bovine placental lactogen, prolactin-related protein-I, and pregnancy-associated glycoproteins. Lectin histochemistry with Dolichos biflorus agglutinin, Vicia villosa agglutinin, and Phaseolus vulgaris leucoagglutinin showed specific staining of BNCs. Different stages of BNC migration and fusion with uterine epithelial cells were observed. Trinucleate feto-maternal hybrid cells were the typical outcome of cell fusions. These cells underwent degeneration, with typical morphological features of apoptosis. The results revealed a strong homology between water buffalo and cattle BNCs concerning cell morphology, protein expression, glycosylation pattern, and characteristics of cell migration and fusion.     

Generation of trophoblast stem cells from rabbit embryonic stem cells with BMP4

Trophoblast stem (TS) cells are ideal models to investigate trophectoderm differentiation and placental development. Herein, we describe the derivation of rabbit trophoblast stem cells from embryonic stem (ES) cells. Rabbit ES cells generated in our laboratory were induced to differentiate in the presence of BMP4 and TS-like cell colonies were isolated and expanded. These cells expressed the molecular markers of mouse TS cells, were able to invade, give rise to derivatives of TS cells, and chimerize placental tissues when injected into blastocysts. The rabbit TS-like cells maintained self-renewal in culture medium with serum but without growth factors or feeder cells, whilst their proliferation and identity were compromised by inhibitors of FGFs and TGF-β receptors. Taken together, our study demonstrated the derivation of rabbit TS cells and suggested the essential roles of FGF and TGF-β signalings in maintenance of rabbit TS cell self-renewal.     

Differential expression of the metastasis suppressor KAI1 in decidual cells and trophoblast giant cells at the feto-maternal interface

Invasion of trophoblasts into maternal uterine tissue is essential for establishing mature feto-maternal circulation. The trophoblast invasion associated with placentation is similar to tumor invasion. In this study, we investigated the role of KAI1, an anti-metastasis factor, at the maternal-fetal interface during placentation. Mouse embryos were obtained from gestational days 5.5 (E5.5) to E13.5. Immunohistochemical analysis revealed that KAI1 was expressed on decidual cells around the track made when a fertilized ovum invaded the endometrium, at days E5.5 and E7.5, and on trophoblast giant cells, along the central maternal artery of the placenta at E9.5. KAI1 in trophoblast giant cells was increased at E11.5, and then decreased at E13.5. Furthermore, KAI1 was upregulated during the forskolinmediated trophoblastic differentiation of BeWo cells. Collectively, these results indicate that KAI1 is differentially expressed in decidual cells and trophoblasts at the maternal-fetal interface, suggesting that KAI1 prevents trophoblast invasion during placentation. [BMB Reports 2013; 46(10): 507-512]