Immunofluorescent localization of a monoclonally defined carbohydrate cell surface antigen (IIC3) during mouse development

A monoclonal antibody (anti-IIC3), raised against F9 embryonal carcinoma cells, detects an antigen which is first expressed at the compacted morula stage and segregates with the trophectoderm of the mouse blastocyst. We have further examined the expression of this antigen during embryonic development. Immunofluorescence experiments on sectioned embryos demonstrate that IIC3 expression is associated with the differentiation of extra-embryonic cell types. It is expressed at the cell surface of the trophectoderm of the attaching blastocyst and differentially by the two derivatives of this layer. The primary and secondary trophoblastic giant cells label intracellularly, whereas the cells of the ectoplacental cone and labyrinth placenta label at the cell surface. IIC3 is also expressed by the primitive endoderm of the blastocyst and subsequently by the visceral endoderm. The parietal endoderm does not express IIC3. Partial characterization of the IIC3 antigen with sugar hapten inhibition and glycosidase digestion experiments, suggests that the antigen is a lactosaminoglycan-like molecule, with galactose and N-acetylgalactosamine residues representing part of the antigenic determinant. Neuraminidase and fucosidase treatment exposed additional anti-IIC3-antigenic sites on the extra-embryonic ectoderm and chorion. A possible role for IIC3 in normal embryonic-uterine interactions is discussed.     

Carbohydrate changes in pre- and peri-implantation mouse embryos as detected by a monoclonal antibody

We have examined the tissue and embryonic distribution of an antigen on a large polysaccharide that is recognized by a monoclonal antibody, IIC3, prepared against F9 teratocarcinoma cells. By immunofluorescence the antigen is first detected on compacted morulae and early blastocysts. It is strongly expressed on the primary endoderm and trophoblast of expanded blastocysts, but then disappears from the trophoblast of attached blastocysts in vitro. The binding of the antibody is completely inhibited by D-galactose and N-acetylgalactosamine. Fluoresceinated lectins were used to study further the changes in cell surface carbohydrates on trophoblast during implantation. Ricinus I, specific for terminal galactose, binds to preimplantation stages but does not bind to the trophoblast of the attached blastocyst. On the other hand, wheat germ agglutinin, specific for N-acetylglucosamine and sialic acid, binds to all preimplantation embryos and also to attached blastocysts (embryo proper and trophoblast). Neuraminidase treatment of blastocyst outgrowths enhances binding of both IIC3 and Ricinus I to the trophoblast; conversely, the binding of wheat germ agglutinin is decreased by this treatment. The results obtained in this study show changes of cell surface carbohydrates during early mouse development and suggest that sialic acid may be masking molecules on the surface of the trophoblast at the time of implantation.     

Analysis of cell surface galactosyltransferase activity during mouse trophectodermal differentiation

The ectoplacental cone (EPC) of the Day 7.5 mouse embryo consists of a core of adhesive, proliferating trophoblast cells which transform to invasive trophoblast giant cells during implantation. Adhesive trophoblast cell types express monoclonally defined lactosaminoglycans (LAGs) at the cell surface; transformation to giant cells results in a loss of LAG cell surface expression (H. J. Hathaway and B. S. Babiarz, 1988, Cell Differ. 24, 55-66). LAGs can serve as substrates for cell surface galactosyltransferase (GalTase), providing an adhesive mechanism between a number of different cell types (B. D. Shur, 1984, Mol. Cell. Biochem. 61, 143-158). It was hypothesized that the LAGs in the EPC represented a substrate for a similar GalTase-mediated cell:cell adhesion system. Cell surface GalTase activity was demonstrated on EPC trophoblast on Day 7.5 of development by the incorporation of galactose from exogenous radiolabeled substrate. In 24- to 48-hr EPC trophoblast cultures the enzyme was localized by immunofluorescence to areas of cell:cell contact. Monolayers of differentiated trophoblast giant cells lacked this labeling pattern. The cell surface glycopeptide substrate for GalTase eluted as a single peak with an apparent molecular mass of 15,000 Da. A portion of this material was sensitive to endo-beta-galactosidase digestion, indicating that it contained a LAG structure. Perturbation of the enzyme:substrate complex in 24- to 48-hr EPC outgrowths, with alpha-lactalbumin, uridine 5'-diphosphogalactose, or anti-GalTase antibody, resulted in the disruption of cell:cell contacts. Differentiation to trophoblast giant cells resulted in a loss of sensitivity to surface GalTase perturbation. The results suggest that adhesive EPC trophoblast cells possess a GalTase-mediated cell:cell adhesion system which is downregulated upon differentiation to invasive trophoblast giant cells.     

Modulation of trophoblast stem cell and giant cell phenotypes: analyses using the Rcho-1 cell model

Trophoblast giant cells are located at the maternal-embryonic interface and have fundamental roles in the invasive and endocrine phenotypes of the rodent placenta. In this report, we describe the experimental modulation of trophoblast stem cell and trophoblast giant cell phenotypes using the Rcho-1 trophoblast cell model. Rcho-1 trophoblast cells can be manipulated to proliferate or differentiate into trophoblast giant cells. Differentiated Rcho-1 trophoblast cells are invasive and possess an endocrine phenotype, including the production of members of the prolactin (PRL) family. Dimethyl sulfoxide (DMSO), a known differentiation-inducing agent, was found to possess profound effects on the in vitro development of trophoblast cells. Exposure to DMSO, at non-toxic concentrations, inhibited trophoblast giant cell differentiation in a dose-dependent manner. These concentrations of DMSO did not significantly affect trophoblast cell proliferation or survival. Trophoblast cells exposed to DMSO exhibited an altered morphology; they were clustered in tightly packed colonies. Trophoblast giant cell formation was disrupted, as was the expression of members of the PRL gene family. The effects of DMSO were reversible. Removal of DMSO resulted in the formation of trophoblast giant cells and expression of the PRL gene family. The phenotype of the DMSO-treated cells was further determined by examining the expression of a battery of genes characteristic of trophoblast stem cells and differentiated trophoblast cell lineages. DMSO treatment had a striking stimulatory effect on eomesodermin expression and a reciprocal inhibitory effect on Hand1 expression. In summary, DMSO reversibly inhibits trophoblast differentiation and induces a quiescent state, which mimics some but not all aspects of the trophoblast stem cell phenotype.     

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.     

SOCS3: an essential regulator of LIF receptor signaling in trophoblast giant cell differentiation

Suppressor of cytokine signaling 3 (SOCS3) binds cytokine receptors and thereby suppresses cytokine signaling. Deletion of SOCS3 causes an embryonic lethality that is rescued by a tetraploid rescue approach, demonstrating an essential role in placental development and a non-essential role in embryo development. Rescued SOCS3-deficient mice show a perinatal lethality with cardiac hypertrophy. SOCS3-deficient placentas have reduced spongiotrophoblasts and increased trophoblast secondary giant cells. Enforced expression of SOCS3 in a trophoblast stem cell line (Rcho-1) suppresses giant cell differentiation. Conversely, SOCS3-deficient trophoblast stem cells differentiate more readily to giant cells in culture, demonstrating that SOCS3 negatively regulates trophoblast giant cell differentiation. Leukemia inhibitory factor (LIF) promotes giant cell differentiation in vitro, and LIF receptor (LIFR) deficiency results in loss of giant cell differentiation in vivo. Finally, LIFR deficiency rescues the SOCS3-deficient placental defect and embryonic lethality. The results establish SOCS3 as an essential regulator of LIFR signaling in trophoblast differentiation.     

The phosphatidylinositol 3-kinase/Akt signaling pathway modulates the endocrine differentiation of trophoblast cells

Activation of Lyn, a Src-related nonreceptor tyrosine kinase, in trophoblast cells is associated with trophoblast giant cell differentiation. The purpose of the present work was to use Lyn as a tool to identify signaling pathways regulating the endocrine differentiation of trophoblast cells. The Src homology 3 domain of Lyn was shown to display differentiation-dependent associations with other regulatory proteins, including phosphatidylinositol 3-kinase (PI3-K). PI3-K activation was dependent upon trophoblast giant cell differentiation. The downstream mediator of PI3-K, Akt/protein kinase B, also exhibited differentiation-dependent activation. Lyn is a potential regulator of the PI3-K/Akt signaling pathway, as are receptor tyrosine kinases. Protein tyrosine kinase profiling was used to identify two candidate regulators of the PI3-K/Akt pathway, fibroblast growth factor receptor-1 and Sky. At least part of the activation of Akt in differentiating trophoblast giant cells involves an autocrine growth arrest-specific-6-Sky signaling pathway. Inhibition of PI3-K activities via treatment with LY294002 disrupted Akt activation and interfered with the endocrine differentiation of trophoblast giant cells. In summary, activation of the PI3-K/Akt signaling pathway regulates the development of the differentiated trophoblast giant cell phenotype.     

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.     

Periodic expression of the cyclin-dependent kinase inhibitor p57(Kip2) in trophoblast giant cells defines a G2-like gap phase of the endocycle

Endoreduplication is an unusual form of cell cycle in which rounds of DNA synthesis repeat in the absence of intervening mitoses. How G1/S cyclin-dependent kinase (Cdk) activity is regulated during the mammalian endocycle is poorly understood. We show here that expression of the G1/S Cdk inhibitor p57(Kip2) is induced coincidentally with the transition to the endocycle in trophoblast giant cells. Kip2 mRNA is constitutively expressed during subsequent endocycles, but the protein level fluctuates. In trophoblast giant cells synchronized for the first few endocycles, the p57(Kip2) protein accumulates only at the end of S-phase and then rapidly disappears a few hours before the onset of the next S-phase. The protein becomes stabilized by mutation of a C-terminal Cdk phosphorylation site. As a consequence, introduction of this stable form of p57(Kip2) into giant cells blocks S-phase entry. These data imply that p57(Kip2) is subject to phosphorylation-dependent turnover. Surprisingly, although this occurs in endoreduplicating giant cells, p57(Kip2) is stable when ectopically expressed in proliferating trophoblast cells, indicating that these cells lack the mechanism for protein targeting and/or degradation. These data show that the appearance of p57(Kip2) punctuates the completion of DNA replication, whereas its turnover is subsequently required to initiate the next round of endoreduplication in trophoblast giant cells. Cyclical expression of a Cdk inhibitor, by terminating G1/S Cdk activity, may help promote the resetting of DNA replication machinery.     

Parp1-deficiency induces differentiation of ES cells into trophoblast derivatives

Embryonic stem (ES) cells deficient in the enzyme poly(ADP-ribose) polymerase (Parp1) develop into teratocarcinoma-like tumors when injected subcutaneously into nude mice that contain cells with giant cell-like morphology. We show here that these cells express genes characteristic of trophoblast giant cells and thus belong to the trophectoderm lineage. In addition, Parp1(-/-) tumors contained other trophoblast subtypes as revealed by expression of spongiotrophoblast-specific marker genes. The extent of giant cell differentiation was enhanced, however, as compared with spongiotrophoblast. A similar shift toward trophoblast giant cell differentiation was observed in cultures of Parp1-deficient ES cells and in placentae of Parp1(-/-) embryos. Analysis of other cell lineage markers demonstrated that Parp1 acts exclusively in trophoblast to suppress differentiation. Surprisingly, trophoblast derivatives were also detected in wildtype tumors and cultured ES cells, albeit at significantly lower frequency. These data show that wildtype ES cells contain a small population of cells with trophectoderm potential and that absence of Parp1 renders ES cells more susceptible to adopting a trophoblast phenotype.     

Giant cell formation in ectopic mouse trophoblast

Segmenting mouse ova, grafted beneath the kidney capsule of syngenic adult recipients, result in a growth of trophoblast, which changes from small, actively-dividing cells into giant trophoblast cells which degenerate 15 days after grafting. Similar giant cells are found in normal mouse placentas. Radioautography with ³H-thymidine, uridine, and leucine revealed cessation of DNA synthesis after day 8, with decline in RNA synthesis from day 10, and continued protein synthesis through day 15. Treatment with Colcemid reduced the graft size but failed to suppress giant cell formation. Treatment on days 4–7 of grafting with 5-fluorodeoxyuridine (FUdR), cyclohexamide, or actinomycin D resulted in giant cell suppression with the maintenance of healthy-appearing small trophoblast cells. These results confirm the early withdrawal of trophoblast grafts from the mitotic pool and the non-mitotic increase of trophoblast DNA, and demonstrate the apparent need for RNA and protein synthesis to support the development of trophoblast giant cells.     

Reprogramming the Cell Cycle for Endoreduplication in Rodent Trophoblast Cells

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34^cdk1 complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.     

FOSL1 is integral to establishing the maternal-fetal interface

Remodeling of uterine spiral arteries by trophoblast cells is a requisite process for hemochorial placentation and successful pregnancy. The rat exhibits deep intrauterine trophoblast invasion and accompanying trophoblast-directed vascular modification. The involvement of phosphatidylinositol 3 kinase (PI3K), AKT, and Fos-like antigen 1 (FOSL1) in regulating invasive trophoblast and hemochorial placentation was investigated using Rcho-1 trophoblast stem cells and rat models. Disruption of PI3K/AKT with small-molecule inhibitors interfered with the differentiation-dependent elaboration of a signature invasive-vascular remodeling trophoblast gene expression profile and trophoblast invasion. AKT isoform-specific knockdown also affected the signature invasive-vascular remodeling trophoblast gene expression profile. Nuclear FOSL1 increased during trophoblast cell differentiation in a PI3K/AKT-dependent manner. Knockdown of FOSL1 disrupted the expression of a subset of genes associated with the invasive-vascular remodeling trophoblast phenotype, including the matrix metallopeptidase 9 gene (Mmp9). FOSL1 was shown to occupy regions of the Mmp9 promoter in trophoblast cells critical for the regulation of Mmp9 gene expression. Inhibition of FOSL1 expression also abrogated trophoblast invasion, as assessed in vitro and following in vivo trophoblast-specific lentivirally delivered FOSL1 short hairpin RNA (shRNA). In summary, FOSL1 is a key downstream effector of the PI3K/AKT signaling pathway responsible for development of trophoblast lineages integral to establishing the maternal-fetal interface.