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.     

Progressive expression of trophoblast-specific genes during formation of mouse trophoblast giant cells in vitro

The expression of a battery of trophoblast-specific mRNAs was studied during trophectoderm development in vivo and in vitro to assess the use of these mRNAs as markers of trophoblast differentiation and to examine lineage relationships between various trophectoderm derivatives. In situ hybridization of sectioned day 6.5–18.5 mouse embryos localized mRNAs for mouse placental lactogens I and II and mouse proliferin (PLF) to trophoblast giant cells and proliferin-related protein mRNA to the spongiotrophoblast and giant cell layers. A fifth marker, cDNA 4311, was found only in spongiotrophoblast. Day 3.5 blastocyst outgrowths and day 7.5 diploid extraembryonic ectoderm (EX) and ectoplacental cone (EPC) were then cultured to produce polyploid giant cells in vitro. Cultures were processed for in situ hybridization after 2, 4, or 6 days. EX and EPC both formed secondary giant cells, which expressed all markers in the same sequence as was observed in vivo, and primary giant cells in blastocyst outgrowths expressed the early giant cell markers PLF and PL-I on days 4 and 6 of culture. EPC progressed through the sequence 2 days ahead of EX, indicating commitment of EPC to giant cell formation. These results suggest that EX, EPC, and primary and secondary giant cells all share in a common pathway of differentiation and that the highly ordered sequence of gene expression characteristic of this pathway occurs similarly in vivo and in vitro. © 1993 Wiley-Liss, Inc.     

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.     

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.     

EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells

Background

The uptake of abortion-inducing pathogens by trophoblast giant (TG) cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70) contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this.

Methods

Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR) domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice.

Results

The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70.

Conclusions

Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.     

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]     

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.     

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.     

An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development

The closely related mitogen-activated protein kinase isoforms extracellular signal-regulated kinase 1 (ERK1) and ERK2 have been implicated in the control of cell proliferation, differentiation and survival. However, the specific in vivo functions of the two ERK isoforms remain to be analysed. Here, we show that disruption of the Erk2 locus leads to embryonic lethality early in mouse development after the implantation stage. Erk2 mutant embryos fail to form the ectoplacental cone and extra-embryonic ectoderm, which give rise to mature trophoblast derivatives in the fetus. Analysis of chimeric embryos showed that Erk2 functions in a cell-autonomous manner during the development of extra-embryonic cell lineages. We also found that both Erk2 and Erk1 are widely expressed throughout early-stage embryos. The inability of Erk1 to compensate for Erk2 function suggests a specific function for Erk2 in normal trophoblast development in the mouse, probably in regulating the proliferation of polar trophectoderm cells.     

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.     

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.     

Syncytins expression in cultured trophoblast cells according to differentiation status

Background  

Data of syncytin 1 and 2 env gene expression in human placenta and participation in the syncytialisation phenomena has been reported. However, there are not many studies on simultaneous changes in expression of both syncytins in culture. We sought evidence on the relative expression of syncytins and syncytin 1 receptors in trophoblast cell culture treated with a differentiation inducing factor (forskolin).     

Lethal giant discs, a novel C2-domain protein, restricts notch activation during endocytosis

The Notch signaling pathway plays a central role in animal growth and patterning, and its deregulation leads to many human diseases, including cancer. Mutations in the tumor suppressor lethal giant discs (lgd) induce strong Notch activation and hyperplastic overgrowth of Drosophila imaginal discs. However, the gene that encodes Lgd and its function in the Notch pathway have not yet been identified. Here, we report that Lgd is a novel, conserved C2-domain protein that regulates Notch receptor trafficking. Notch accumulates on early endosomes in lgd mutant cells and signals in a ligand-independent manner. This phenotype is similar to that seen when cells lose endosomal-pathway components such as Erupted and Vps25. Interestingly, Notch activation in lgd mutant cells requires the early endosomal component Hrs, indicating that Hrs is epistatic to Lgd. These data suggest that Lgd affects Notch trafficking between the actions of Hrs and the late endosomal component Vps25. Taken together, our data identify Lgd as a novel tumor-suppressor protein that regulates Notch signaling by targeting Notch for degradation or recycling.     

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.     

Cyclins E1 and E2 are required for endoreplication in placental trophoblast giant cells

In mammalian cells, cyclin E-CDK2 complexes are activated in the late G1 phase of the cell cycle and are believed to have an essential role in promoting S-phase entry. We have targeted the murine genes CCNE1 and CCNE2, encoding cyclins E1 and E2. Whereas single knockout mice were viable, double knockout embryos died around midgestation. Strikingly, however, these embryos showed no overt defects in cell proliferation. Instead, we observed developmental phenotypes consistent with placental dysfunction. Mutant placentas had an overall normal structure, but the nuclei of trophoblast giant cells, which normally undergo endoreplication and reach elevated ploidies, showed a marked reduction in DNA content. We derived trophoblast stem cells from double knockout E3.5 blastocysts. These cells retained the ability to differentiate into giant cells in vitro, but were unable to undergo multiple rounds of DNA synthesis, demonstrating that the lack of endoreplication was a cell-autonomous defect. Thus, during embryonic development, the needs for E-type cyclins can be overcome in mitotic cycles but not in endoreplicating cells.