Primary Trophoblast Giant Cells Research Articles

Trophoblastic signals facilitate endometrial interferon response and lipid metabolism, ensuring normal decidualization

The decidua plays a crucial role in providing structural and trophic support to the developing conceptus before placentation. Following embryo attachment, embryonic components intimately interact with the decidual tissue. While evidence indicates the participation of embryo-derived factors in crosstalk with the uterus, the extent of their impact on post-implantation decidual development requires further investigation. Here, we utilize transgenic mouse models to selectively eliminate primary trophoblast giant cells (pTGCs), the embryonic cells that interface with maternal tissue at the forefront. pTGC ablation impairs decidualization and compromises decidual interferon response and lipid metabolism. Mechanistically, pTGCs release factors such as interferon kappa (IFNK) to strengthen the decidual interferon response and lipoprotein lipase (LPL) to enhance lipid accumulation within the decidua, thereby promoting decidualization. This study presents genetic and metabolomic evidence reinforcing the proactive role of pTGC-derived factors in mobilizing maternal resources to strengthen decidualization, facilitating the normal progression of early pregnancy.

Dynamic Changes of Gene Expression in Mouse Mural Trophectoderm Regulated by Cdx2 During Implantation.

Implantation of the blastocyst into the uterus is a specific and essential process for mammalian embryonic development. In mice, implantation is initiated from the mural trophectoderm of the blastocyst and the mTE controls implantation progression by acquiring the ability to attach and invade into the endometrium while differentiating into primary trophoblast giant cells. Nevertheless, it remains largely unclear when and how the mTE differentiates and acquires this ability during implantation. Here, by RNA sequencing analysis with the pre- and peri-implantation mTE, we show that the mTE undergoes stage-specific and dynamic changes of gene expression during implantation. We also reveal that the mTE begins down-regulating Cdx2 and up-regulating differentiation marker genes during the peri-implantation stage. In addition, using trophectoderm (TE) -specific lentiviral vector-mediated gene transduction, we demonstrate that TE-specific Cdx2 overexpression represses differentiation of the mTE into the primary trophoblast giant cells. Moreover, we reveal that TE-specific Cdx2 overexpression also represses the up-regulation of cell adhesion- and migration-related genes, including Slc6a14, Slc16a3, Itga7, Itgav and Itgb3, which are known to regulate migration of trophectoderm cells. In particular, the expression of Itgb3, an integrin subunit gene, exhibits high inverse correlation with that of Cdx2 in the TE. Reflecting the down-regulation of the genes for TE migration, TE-specific Cdx2 overexpression causes suppression of the blastocyst outgrowth in vitro and abnormal progression of implantation in vivo. Thus, our results specify the time-course changes of global gene expression in the mTE during implantation and uncover the significance of Cdx2 down-regulation for implantation progression.

Persistent DNA Damage and Senescence in the Placenta Impacts Developmental Outcomes of Embryos.

Cohesin is an evolutionarily conserved chromosome-associated protein complex essential for chromosome segregation, gene expression, and repair of DNA damage. Mutations that affect this complex cause the human developmental disorder Cornelia de Lange syndrome (CdLS), thought to arise from defective embryonic transcription. We establish a significant role for placental defects in the development of CdLS mouse embryos (Nipbl and Hdac8). Placenta is a naturally senescent tissue; we demonstrate that persistent DNA damage potentiates senescence and activates cytokine signaling. Mutant embryo developmental outcomes are significantly improved in the context of a wild-type placenta or by genetically restricting cytokine signaling. Our study highlights that cohesin is required for maintaining ploidy and the repair of spontaneous DNA damage in placental cells, suggesting that genotoxic stress and ensuing placental senescence and cytokine production could represent a broad theme in embryo health and viability.

Plac1 Expression Pattern at the Mouse Fetomaternal Interface and Involvement in Trophoblast Differentiation

Background/Aims: It is well known that Plac1 is a placenta-specific gene; however, its spatiotemporal expression pattern and exact role at t h e mouse fetomaternal interface r e m a i n s unclear. Methods: In situ hybridization (ISH) was used to localize the Plac1 mRNA at the mouse fetomaternal interface. A trophoblast stem cell (TS) differentiation model with Plac1 shRNA-overexpressing lentivirus was employed to investigate the possible role of Plac1 in placentation. Real-time RT-PCR was used to detect changes in gene expression. Results: Plac1 was exclusively expressed in the ectoplacental cone (EPC) as well as in 8.5 and 9.5 days post-coitum (dpc) embryos. Subsequently, Plac1 expression was abundant in the spongiotrophoblast layer and moderately in the labyrinth layer until 13.5 dpc, and declined thereafter. Interestingly, Plac1 was also expressed by secondary trophoblast giant cells and glycogen trophoblast cells, but not in primary trophoblast giant cells. Plac1 transcription was increased during the TS differentiation (P < 0.01), and knockdown of Plac1 significantly impaired TS differentiation. Conclusion: Plac1 is abundantly expressed at the fetomaternal interface and in all trophoblast subtypes except in primary trophoblast giant cells. Plac1 knockdown retarded the progress of TS differentiation, indicating that Plac1 is necessary for normal trophoblast differentiation into various trophoblast subpopulations.

Transcriptional Analysis by Nascent RNA FISH of &lt;em&gt;In Vivo&lt;/em&gt; Trophoblast Giant Cells or &lt;em&gt;In Vitro&lt;/em&gt; Short-term Cultures of Ectoplacental Cone Explants

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells differentiate into primary trophoblast giant cells (TGCs) while the polar trophectoderm overlying the inner cell mass continues to proliferate later differentiating into secondary TGCs. TGCs play a key role in developing placenta and are essential for a successful pregnancy. Investigation of transcriptional regulation of specific genes during post-implantation development can give insights into TGCs development. Cells of the ectoplacental cone (EPC) from embryos at 7-7.5 days of gestation (E7-7.5), derived from the polar trophectoderm, differentiate into secondary TGCs1. TGCs can be studied in situ, on cryostat sections of embryos at E7 although the number of TGCs is very low at this stage. An alternative means of analyzing secondary TGCs is to use short-term cultures of individual EPCs from E7 embryos. We propose a technique to investigate the transcriptional status of genes of interest both in vivo and in vitro at the single-cell level using fluorescent in situ hybridization (RNA FISH) to visualize nascent transcripts. This technique provides a direct readout of gene expression and enables assessment of the chromosomal status of TGCs, which are large endoreplicating cells. Indeed, a key feature of terminal differentiation of TGCs is that they exit the cell cycle and undergo multiple rounds of endoreplication.This approach can be applied to detect expression of any gene expressed from autosomes and/or sex chromosomes and can provide important information into developmental mechanisms as well as placental diseases.

Transcriptional Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants.

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells differentiate into primary trophoblast giant cells (TGCs) while the polar trophectoderm overlying the inner cell mass continues to proliferate later differentiating into secondary TGCs. TGCs play a key role in developing placenta and are essential for a successful pregnancy. Investigation of transcriptional regulation of specific genes during post-implantation development can give insights into TGCs development. Cells of the ectoplacental cone (EPC) from embryos at 7-7.5 days of gestation (E7-7.5), derived from the polar trophectoderm, differentiate into secondary TGCs1. TGCs can be studied in situ, on cryostat sections of embryos at E7 although the number of TGCs is very low at this stage. An alternative means of analyzing secondary TGCs is to use short-term cultures of individual EPCs from E7 embryos. We propose a technique to investigate the transcriptional status of genes of interest both in vivo and in vitro at the single-cell level using fluorescent in situ hybridization (RNA FISH) to visualize nascent transcripts. This technique provides a direct readout of gene expression and enables assessment of the chromosomal status of TGCs, which are large endoreplicating cells. Indeed, a key feature of terminal differentiation of TGCs is that they exit the cell cycle and undergo multiple rounds of endoreplication.This approach can be applied to detect expression of any gene expressed from autosomes and/or sex chromosomes and can provide important information into developmental mechanisms as well as placental diseases.

Phosphoinositide 3-Kinase (PI3K) Subunit p110δ Is Essential for Trophoblast Cell Differentiation and Placental Development in Mouse.

Maternal PI3K p110δ has been implicated in smaller litter sizes in mice, but its underlying mechanism remains unclear. The placenta is an indispensable chimeric organ that supports mammalian embryonic development. Using a mouse model of genetic inactivation of PI3K p110δ (p110δD910A/D910A), we show that fetuses carried by p110δD910A/D910A females were growth retarded and showed increased mortality in utero mainly during placentation. The placentas in p110δD910A/D910A females were anomalously anemic, exhibited thinner spongiotrophoblast layer and looser labyrinth zone, which indicate defective placental vasculogenesis. In addition, p110δ was detected in primary trophoblast giant cells (P-TGC) at early placentation. Maternal PI3K p110δ inactivation affected normal TGCs generation and expansion, impeded the branching of chorioallantoic placenta but enhanced the expression of matrix metalloproteinases (MMP-2, MMP-12). Poor vasculature support for the developing fetoplacental unit resulted in fetal death or gross growth retardation. These data, taken together, provide the first in vivo evidence that p110δ may play an important role in placental vascularization through manipulating trophoblast giant cell.

Determination of Allelic Expression of H19 in Peri-Implantation Mouse Embryos.

H19 is a maternally-expressed imprinted non-coding RNA with tumor suppressor activity. During mouse preimplantation development, H19 is primarily expressed in the trophectoderm cells. It has been documented that preimplantation embryo manipulation affects the allelic expression of this gene causing it to become biallelically expressed. We have observed biallelic expression of H19 in embryos that developed in vivo leading us to hypothesize that H19 biallelic expression might be a normal occurrence in mouse embryos immediately prior to implantation. Expression of H19 was determined in C7xB6 F1 embryos derived from superovulated C57BL/6J(Cast7) (C7) females mated to C57BL/6J (B6) males. In vivo-produced control embryos were collected from the uterus. In vitro-cultured embryos were collected from the oviduct at the 2-cell stage and cultured in KSOM supplemented with amino acids or Whitten's media. The embryo collection times chosen were 84, 96, and 108 hours post presumed ovulation to obtain embryos at the early, mid, and late blastocyst stages of development. QPCR with subsequent FRET or RFLP followed by PAGE allowed us to determine allele-specific gene expression in single embryos. For an embryo to be considered to have biallelic H19 expression, =10% of the total H19 RNA must be paternal in origin. So far we have analyzed H19 expression in 21, 32, 47 in vivo produced embryos (for 84, 96, 108 h; respectively), 18, 52, 58 KSOM cultured embryos, and 11, 44, 31 Whitten's cultured embryos. Those results show that the paternal allele is expressed in 24,31, 77 % of in vivo produced embryos (for 84, 96, 108 h respectively), 28, 31, 60 % of KSOM cultured embryos and 18, 15, 55 % of Whitten's cultured embryos. As recent reports suggest a possible effect of superovulation on imprinting, we are repeating the experiment with embryos produced after natural ovulation. Thus far we have analyzed H19 expression in 5, 25, 15 in vivo produced embryos (for 84, 96, 108 h, respectively), 1, 19, 25 KSOM cultured embryos and 0, 8, 20 Whitten's cultured embryos. Those results show that the paternal allele is expressed in 20, 32, 80 % of in vivo produced embryos (for 84, 96, 108 h respectively), 0, 32, 60 % of KSOM culture embryos and not detected, 25, 30 % of Whitten's cultured embryos. We are currently performing DNA methylation assays to ascertain if the observed loss of imprinting correlates with a concomitant decrease in DNA methylation at the H19 regulatory region. Peri-implantation mouse embryos are composed of at least five cell types, namely, inner cell mass, primitive endoderm, mural trophectoderm, polar trophectoderm, and trophoblast giant cells. We speculate that biallelic H19 expression is the result of the differentiation of the trophectoderm trophoblast giant cells and that this may be relevant for implantation. We are currently performing microdissection studies to divide the embryo in two-three parts, one containing the inner cell mass cells, primitive endoderm cells and/or adjacent polar trophectoderm and the other containing the mural trophectoderm and primary trophoblast giant cells. Using a combination of TaqMan probes for H19, Igf2, Pou5f1, Cdx2, Rdx, Dek we intend to ascertain the enrichment of giant cells in our mural trophectoderm sample. Initial work in this area demonstrates our ability to have mural trophectoderm cells free or with minimal inner cell mass contamination based on lack of expression of Pou5f1 in this embryo section.

Indoleamine 2,3-dioxygenase expression is restricted to fetal trophoblast giant cells during murine gestation and is maternal genome specific

Pharmacologic inhibition of indoleamine 2,3 dioxygenase (IDO) activity during murine pregnancy results in rejection of allogeneic fetuses by the maternal immune system. Here, we show that IDO expression is restricted to perinuclear regions of primary trophoblast giant cells (TGCs) of fetal origin at mid-gestation (E10.5). After placentation (E14), no IDO expression was detected at the maternal–fetal interface. Matings involving IDO-deficient females revealed that paternally inherited IDO alleles were inactive in primary TGCs, presumably due to paternal genome-specific gene inactivation. Allogeneic matings in which both parents were genetically IDO-deficient produced litters of normal sizes at normal rates compared to IDO-sufficient parental mice, implying that compensatory or redundant immunosuppressive mechanisms protected allogeneic fetuses during gestation in IDO-deficient mice. Consistent with this notion, treatment with IDO inhibitor did not affect allogeneic pregnancy rates when both parents were IDO-deficient, confirming that IDO was the relevant pharmacologic target of the IDO inhibitor in matings involving IDO-sufficient mice. Hence, IDO is a key immunosuppressive mechanism in normal murine pregnancies, and it is regulated entirely through maternally inherited fetal genes.

Immunohistochemical analysis of protein gene product 9.5, a ubiquitin carboxyl-terminal hydrolase, during placental and embryonic development in the mouse.

Protein gene product 9.5 (PGP9.5) is expressed at high level in the neural and neuroendocrine systems. We investigated the localization and degree of expression of PGP9.5 in the developing mouse placenta and embryo at 6.5, 10.5 and 14 days of gestation using an immunohistochemical technique. At 6.5 days of gestation PGP9.5 was detected at various levels in decidual and primary trophoblast giant cells in the placenta, and in embryonic ectodermal cells in the embryo. At 10.5 and 14 days of gestation PGP9.5 was expressed at moderate to strong levels in neurons in the embryo, but rarely in the placenta. These findings suggest that the protein may play a significant role in implantation and placental development, and differentiation of embryonic ectoderm.

Effects of altered gene expression on ES cell differentiation.

AbstractLittle is known about the molecular mechanisms controlling the earliest cellular differentiation events of the mammalian embryo. Pluripotent embryonic stem (ES) cell lines, derived from cells of the inner cell mass of the blastocyst, are an important experimental system that can be used to study the differentiation of the mammalian embryo into its earliest recognizable tissue lineages. For instance, experiments performed in ES cells demonstrate the essential role of the homeobox-containing gene Oct3/4 in the totipotent cells of the preimplantation embryo (1). When two alleles of Oct3/4 are mutated in ES cells, Oct3/4-null cells lose their pluripotency and differentiate into only trophoblast cells. In contrast, when Oct3/4 is overexpressed in ES cells, spontaneous differentiation occurs and a variety of mesoderm-specific and extraembryonic endoderm-specific genes are expressed. Similar results have been obtained from in vivo experiments using Oct3/4 knock-out mice (2). Mouse preimplantation embryos, with two null alleles of Oct3/4, develop into blastocysts containing primary trophoblast giant cells but lacking inner cell mass cells. Taken together, the in vitro and in vivo studies of Oct3/4 function indicate that Oct3/4 is one of the key factors regulating the differentiation of totipotent cells of the preimplantation embryo.KeywordsEmbryonic Stem CellLeukemia Inhibitory FactorEmbryoid BodyPreimplantation EmbryoRibonuclease Protection AssayThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Expression of cathepsin proteinases by mouse trophoblast in vivo and in vitro.

Implantation and placentation in the mouse requires successful invasion of the uterine wall by primary and secondary trophoblast giant cells. Their invasive nature depends in part on the upregulation of proteinases for the phagocytosis and extracellular digestion of maternal cells and matrix materials. The work reported here studies the expression of cathepsin proteinases during secondary trophoblast differentiation, and compares the expression patterns to fully differentiated day 8.5 primary trophoblast giant cells. Cathepsins B (CB), L (CL), and D (CD) were found to be upregulated during trophoblast differentiation in vivo at the message and protein level producing expression patterns equivalent to those of primary trophoblast. Invasive trophoblast cells expressed higher levels of the processed or active forms of the enzymes, coinciding with the period of trophoblast phagocytosis of maternal blood, decidual cells, and matrix materials. Trophoblast differentiation in vitro showed a similar upregulation of cathepsin enzymes. The enzymes were localized to heterogeneous vesicles that resembled both lysosomes and heterophagic vesicles. The presence of a large lysosomal population within the giant cells was confirmed by vital staining with acridine orange. Analysis of trophoblast-conditioned media also demonstrated secreted forms of CB and CL. The results suggest that cathepsin enzymes may contribute to trophoblast invasion not only through the intracellular breakdown of molecules phagocytosed by trophoblast cells, but also by the extracellular digestion of matrix molecules and activation of other pro-enzymes.

Lack of coupling between onset of giant transformation and genome endoreduplication in the mural trophectoderm of the mouse blastocyst

Prominent among the various types of cell that differentiate from the trophectoderm of the mouse blastocyst are trophoblastic giant cells. Repeated endoreduplication of the genome accompanies the growth of these cells, which have been shown to be polytene rather than polyploid. Early stages in giant transformation have been examined, mainly in the mural trophectoderm of the implanting blastocyst which gives rise to the primary trophoblastic giant cells. One confusing issue is whether these early stages include the onset of endoreduplication of the genome. This issue has been addressed in the present study by comparing the DNA content of nuclei in isolated trophectoderm and ICM tissue rather than, as previously, by relating measurements on air-dry preparations of entire blastocysts to those of adult liver. The results, particularly those from delayed and reactivated blastocysts, show that genome endoreduplication is not an obligatory early event in the transformation of mural trophectoderm cells.

Class I major histocompatibility complex antigen expression on early murine trophoblast and its induction by lymphokines in vitro. II. The role of gamma interferon in the responses of primary and secondary giant cells

The induction of paternal Class I and II MHC antigens by crude lymphokine preparations or purified recombinant gamma interferon was investigated on (C57BL/6 J × CBA/ H)F 1 primary and secondary trophoblast giant cell outgrowths from 3.5-day post-coital (pc) blastocyst and 7.5-day pc ectoplacental cone preparations, respectively, using sensitive immunogold labelling techniques and electron microscopy. Class I MHC (but not Class II) antigens could readily be induced on secondary trophoblast giant cells, by incubation in vitro with gamma interferon for 40 h. However, repeated attempts to induce detectable MHC antigens on primary trophoblast giant cells failed. Mock-treated (C57BL/6 J × CBA/ H)F 1 secondary trophoblast giant cell control preparations failed to express detectable MHC antigens. These findings suggest that, at the time of implantation, there is a time window during which MHC antigens are neither expressed constitutively nor are inducible by soluble factors which normally modulate cell surface MHC antigen concentration.

Developmentally regulated expression of the cell-cell adhesion glycoprotein cell-CAM [formula omitted] in peri-implantation mouse embryos and extraembryonic membranes

Peri-implantation mouse embryos and extraembryonic membranes were examined immunohistochemically for the expression of the cell-cell adhesion molecule (cell-CAM) 120 80 . Cell-CAM 120 80 was seen along the lateral borders of all cells in the blastocyst but became undetectable on trophoblastic giant cells, some mononuclear trophoblastic cells and parietal yolk sac cells when blastocysts were cultured in vitro. In postimplantation embryos in vivo, all parts of the early egg-cylinder reacted with the antibody to cell-CAM 120 80 except for the cells of the parietal endoderm and the primary trophoblastic giant cells. In the late stage egg-cylinder, no cell-CAM 120 80 was seen on the cells of the primitive mesoderm or on the primordial germ cells. The germ cells in genital ridges and fetal gonads remained cell-CAM 120 80 - negative throughout the fetal stages of development. In the extraembryonic membranes, the visceral yolk sac, amnion, and the cells of the placental labyrinth were cell-CAM 120 80 - positive , whereas, the parietal yolk sac cells and the spongiotrophoblast cells were negative. These data show that cell-CAM 120 80 is found on cells arranged into epithelial layers in the early embryo and extraembryonic tissues, but is not expressed in the dissociated cells differentiating from these epithelia. Thus, the expression of cell-CAM 120 80 appears to be developmentally regulated.

Binucleate cells in mouse morulae

Mouse morulae from two strains were examined in whole mounts after dissociation of embryos into single cells and were analysed in serial sections by light and electron microscopy. One or two binucleate cells per embryo were discovered in a statistically significant number of morulae. The frequency of morulae with binucleate cell(s) was higher in older morulae than in younger ones. Binucleate cells were always the outer cells of the embryo. Their ultrastructure did not differ from the ultrastructure of mononucleate cells. It is suggested that cell binuclearity at the morula stage is a possible way to polyploidization of nuclei, resulting in the formation of primary trophoblast giant cells.

Placental polyploidization: a comparative study in the mouse and the guinea pig

The trophoblastic and endodermal tissues of the guinea pig were cytophotometrically examined at two stages of development, either shortly after the primary trophoblastic giant cell layer had degenerated (Sansom and Hill, 1931) and the visceral yolk sac was starting to form (Kaufman and Davidoff, 1977) (c. 10.5 days p.c. at the egg cylinder stage) or later in gestation, when the yolk sac was well developed (c. 17.5 days p.c. at the neural tub closure stage). Subsequently, the trophoblastic and endodermal tissues of the mouse were analysed in a manner identical to those of the guinea pig. Finally, the nuclear DNA content values from the analysis of the tissues of each species were compared. The results thus obtained indicate firstly that, in the guinea pig, polyploid nuclei also appear within visceral endoderm after the primary trophoblastic giant cells degenerate and, secondly, that placental specialization in the rodents, as originally defined by Mossman (1937), apparently tends towards lower levels of polyploidy within trophoblast and higher levels within the derivatives of visceral endoderm.

The expression of surface antigens on three trophoblastic tissues in the mouse.

The expression of paternal and maternal antigens on three types of mouse trophoblast cultured in vitro has been examined with the mixed haemadsorption test. Primary trophoblastic giant cells outgrown from blastocysts and placental trophoblast from 16-day pregnancies expressed antigens of both sources at all stages in culture. Secondary trophoblastic giant cells outgrown from the tip of 8-day ectoplacental cones did not give a positive reaction until they had outgrown for 3-5 days. Microdensitometry after Feulgen staining showed that the cells which gave a positive mixed haemadsorption response were polyploid. Incubation of blastocysts and ectoplacental cone tissue with progesterone, oestradiol-17beta and hCG did not affect the results.

Implantation of the rat egg. III. Origin and development of primary trophoblast giant cells

Implantation of the rat egg. III. Origin and development of primary trophoblast giant cells