Subpopulations Of Trophoblast Cells Research Articles

Single-cell and spatial transcriptomics reveal alterations in trophoblasts at invasion sites and disturbed myometrial immune microenvironment in placenta accreta spectrum disorders

BackgroundPlacenta accreta spectrum disorders (PAS) are a severe complication characterized by abnormal trophoblast invasion into the myometrium. The underlying mechanisms of PAS involve a complex interplay of various cell types and molecular pathways. Despite its significance, both the characteristics and intricate mechanisms of this condition remain poorly understood.MethodsSpatial transcriptomics (ST) and single-cell RNA sequencing (scRNA-seq), were performed on the tissue samples from four PAS patients, including invasive tissues (ST, n = 3; scRNA-seq, n = 4), non-invasive normal placenta samples (ST, n = 1; scRNA-seq, n = 2). Three healthy term pregnant women provided normal myometrium samples (ST, n = 1; scRNA-seq, n = 2). ST analysis characterized the spatial expression landscape, and scRNA-seq was used to identify specific cellular components in PAS. Immunofluorescence staining was conducted to validate the findings.ResultsST slices distinctly showed the myometrium in PAS was invaded by three subpopulations of trophoblast cells, extravillous trophoblast cells, cytotrophoblasts, and syncytiotrophoblasts, especially extravillous trophoblast cells. The pathways enriched by genes in trophoblasts, smooth muscle cells (SMC), and immune cells of PAS were mainly associated with immune and inflammation. We identified elevated expression of the angiogenesis-stimulating gene PTK2, alongside the cell proliferation-enhancing gene EGFR, within the trophoblasts of PAS group. Trophoblasts mainly contributed the enhancement of HLA-G and EBI3 signaling, which is crucial in establishing immune escape. Meanwhile, SMC regions in PAS exhibited upregulation of immunomodulatory markers such as CD274, HAVCR2, and IDO1, with CD274 expression experimentally verified to be increased in the invasive SMC areas of the PAS group.ConclusionsThis study provided information of cellular composition and spatial organization in PAS at single-cell and spatial level. The dysregulated expression of genes in PAS revealed a complex interplay between enhanced immune escape in trophoblasts and immune tolerance in SMCs during invasion in PAS. These findings will enhance our understanding of PAS pathogenesis for developing potential therapeutic strategies.

Spatially resolved transcriptomic profiling of placental development in dairy cow.

The placenta plays a crucial role in successful mammalian reproduction. Ruminant animals possess a semi-invasive placenta characterized by a highly vascularized structure formed by maternal endometrial caruncles and fetal placental cotyledons, essential for full-term fetal development. The cow placenta harbors at least two trophoblast cell populations: uninucleate (UNC) and binucleate (BNC) cells. However, the limited capacity to elucidate the transcriptomic dynamics of the placental natural environment has resulted in a poor understanding of both the molecular and cellular interactions between trophoblast cells and niches, and the molecular mechanisms governing trophoblast differentiation and functionalization. To fill this knowledge gap, we employed Stereo-seq to map spatial gene expression patterns at near single-cell resolution in the cow placenta at 90 and 130 days of gestation, attaining high-resolution, spatially resolved gene expression profiles. Based on clustering and cell marker gene expression analyses, key transcription factors, including YBX1 and NPAS2, were shown to regulate the heterogeneity of trophoblast cell subpopulations. Cell communication and trajectory analysis provided a framework for understanding cell-cell interactions and the differentiation of trophoblasts into BNCs in the placental microenvironment. Differential analysis of cell trajectories identified a set of genes involved in regulation of trophoblast differentiation. Additionally, spatial modules and co-variant genes that help shape specific tissue structures were identified. Together, these findings provide foundational insights into important biological pathways critical to the placental development and function in cows.

Immunomodulatory placental exosomes in pregnancy

The placenta is an organ derived at embryogenesis from the trophectoderm and inner cell mass of the blastocyst. Several subpopulations of trophoblast cells, which originate from the trophectoderm, collectively give rise to a “trophoblast shell” that completely encases the fetus for the duration of pregnancy. As such, these cells form the anatomical interface between mother and fetus, and must arbitrate immunological responses of the mother to specific fetal antigens.Antigensexpressed in theplacenta include paternally-inherited as well as placenta-specific antigens; the latter includes at least one antigen, Trophoblast Glycoprotein (TPBG or 5T4) that can later appear as a tumor associated antigen. These antigens access the maternal lymphoid organs, as can be demonstrated immunohistologically, via a hematogenous route. Further, association of fetal/placental antigens with microvesicles and exosomes allows for immune modulation by trophoblast immune modulators co-expressed within these vesicles, which include inhibitory members of the B7 costimulatory family as well as HLA-G. Thus, the adaptive immune response to paternallyinherited and placenta-specific antigens is potently modified by placenta-derived vesicles. Supported by NIH grants HD049480 and HD045611.

Modeling Preeclampsia: An Emerging Role for Stem Cells

Preeclampsia is specific to human pregnancy and is cured by delivery of the gestation. The disease is particularly difficult to study because its underpinnings likely occur very early in pregnancy, but its detection is delayed until the second or third trimester. In vivo study of disease pathogenesis is limited by ethical prohibitions and logistical limitations to our access to the earliest events in human gestation, some of which occur before the first missed menses and clinical detection of the pregnancy. Because the disease does not appear to spontaneously occur in any readily available animal model, study of the disorder in animals is limited by models that mimic some, but not all, of the disease manifestations. The disease may be largely the result of abnormalities in placentation, so human trophoblast cells have been used to allow in vitro study of the disorder. Unfortunately, primary cell lines are typically short-lived while immortalized, or cancer-derived cell lines may carry genetic or epigenetic changes that alter cellular function or morphologic features in ways that may confound result interpretation. Recent advances in stem cell technologies provide a new way to study preeclampsia via accessing the earliest events in placental development. These advances include protocols that promote the conversion of totipotent human embryonic stem cells into trophoblast cells that can differentiate into typical trophoblast cell subpopulations in vitro and the use of induced pluripotent stem cells. The latter can be derived from the gestational tissues of affected and unaffected pregnancies. They may also retain etiologic genetic and epigenetic changes.

The Role of Apoptosis on Trophoblast Cell Invasion in the Placental Bed of Normotensive and Preeclamptic Pregnancies

Background. Placental development depends on careful coordination of trophoblast proliferation and apoptosis; however, the synchrony of its effect on trophoblast invasion is unknown. Objective. To examine the relationship between trophoblast apoptosis and proliferation in placental bed tissue of preeclamptic and normotensive pregnancies. Methods. Serial sections from archived placental bed biopsies of 12 normotensive (group 1) and 12 preeclamptic (group 2) were immunolabeled with a rabbit anti-Ki67 antibody, a mouse anti-cytokeratin 18 and its neo-epitope, and a monoclonal cytodeath M30 antibody. Results. The immunoexpression of Ki67 for all trophoblast cell subpopulations within the myometrium was non-reactive in both study groups. Smooth muscle cells of the microvasculature reflected a moderate degree of proliferation in both groups. Morphometric image analysis of the wall of the spiral artery revealed a mean area of 31,1729 ± 51,180 µm2 compared to 35,795 ± 8045 µm2 in groups 1 and 2, respectively. An elevation of intramural trophoblast was evident within the spiral artery of group 1 (13%). Comparative analyses of M30 distribution on corresponding serial sections were 0.06% versus 0% in groups 1 and 2, respectively. The mean field area percentage of interstitial trophoblast invasion was 10.79% versus 2.87% with corresponding areas of apoptosis been 0.8 % versus 1.9 % in groups 1 and 2, respectively. Conclusions. This study demonstrates an increased trophoblast apoptosis in placental bed of preeclamptic compared to normotensive pregnancies with concurrent absence of proliferation at term.

Assessment of interferon responses in intact human placental explants using a novel whole mount immunofluorescence technique (P3205)

Abstract Pro-inflammatory cytokines required for immunity against non-self, such as interferons (IFNs), are expressed at the maternal-fetal interface during normal pregnancy. However, neither the placental cell targets nor the functions of these cytokines have been defined. We hypothesized that semi-allogeneic trophoblast cells (TBCs), which are the only cells derived from the fertilized egg that are in direct contact with maternal tissue, are hypo-responsive to IFNs in order to evade maternal immune recognition. Therefore, we developed a novel whole mount immunofluorescence technique to identify cellular targets of IFN-γ and IFN-α/β by examining expression of phosphorylated STAT-1 (pSTAT-1) in intact human placental explants. Using this technique we were able to identify the major cell populations present in the placental explants, including fetal macrophages and distinct subpopulations of TBCs. Neither the syncytiotrophoblast layer in direct contact with maternal blood, nor the underlying cytotrophoblast cells expressed pSTAT-1 in response to either IFN. In contrast, extravillous trophoblast cells, which are in direct contact with maternal decidual tissue, and fetal macrophages induced strong expression of pSTAT-1 in response to IFNs. The identification of differential IFN responsiveness in distinct human trophoblast cell subpopulations provides important new insights into the roles of IFNs in normal pregnancy and placental pathology and infection.

HLA-G and pregnancy adverse outcomes

There is growing evidence that pregnancy complications such as preeclampsia, recurrent pregnancy loss (RPL), IUGR, and premature birth could be associated with abnormal immunologic interactions at the fetal–maternal interface. The restricted expression of HLA-G to the subpopulation of trophoblast cells which invade the uterus has generated much interest. The alternative splicing of HLA-G primary transcript, gives origin to seven isoforms, including both membrane-bound forms (HLA-G1, G2, G3, G4) and soluble forms (sHLA-G: sHLA-G5, G6, G7). sHLA-G consists predominantly of sHLA-G1 after its shedding by metalloproteinases, and secreted sHLA-G5 representing the quantitatively dominating and full-length isoforms. HLA-G expression and HLA-G genetic variations in both the mother and the embryo/fetus may be important for pregnancy outcome. It is also intuitively apparent that a gene with putative immunosuppressive and immunotolerant potential might be functional in both the mother and the embryo/fetus/placenta. Reduced or aberrant HLA-G expression seems to be associated with certain complications of pregnancy, among which preeclampsia and possibly the risk of miscarriage, and that this may be further linked to HLA-G polymorphisms. Most of the studies aimed at assessing the role of HLA-G in pregnant diseases have considered only the maternal genotype and ignored the contribution of the fetus. In this regard, the mother, placenta and the fetus form a synthesis. Therefore, studies on placental diseases should address HLA-G expression and genetic variations also to the fetus/placenta.

Placental cell expression of HLA-G2 isoforms is limited to the invasive trophoblast phenotype.

The HLA-G message is alternatively spliced into multiple transcripts, two of which encode soluble isoforms. To initiate studies on the specific functions of the soluble isoforms, we produced soluble rHLA-G1 (rsG1) and rsG2 in human embryonic kidney 293 cells and characterized the proteins. Both isoforms were glycosylated and formed disulfide-bonded oligomers. Recombinant sG1 associated with beta(2)-microglobulin, whereas rsG2 did not. Mouse mAb generated to rsG1 (1-2C3), which identified exclusively sG1, and mAb generated to rsG2 (26-2H11), which identified both soluble and membrane G2 (m/sG2), were used for immunohistochemical isoform mapping studies on placental tissue sections. Soluble G1 protein was abundant in many subpopulations of trophoblast cells, whereas m/sG2 protein was present exclusively in extravillous cytotrophoblast cells. Although both isolated placental villous cytotrophoblast cells and chorion membrane extravillous cytotrophoblast cells contained mRNAs encoding sG1 and sG2, protein expression was as predicted from the immunostains with m/sG2 present only in the invasive trophoblast subpopulation. Analysis of function by Northern and Western blotting demonstrated that both rsG1 and rsG2 inhibit CD8alpha expression on PBMC without changing CD3delta expression or causing apoptotic cell death. Collectively, the studies indicate that: 1) both sG1 and m/sG2 are produced in placentas; 2) transcription and translation are linked for sG1, but not G2; 3) expression of G2 is exclusively associated with the invasive phenotype; and 4) the two isoforms of sG may promote semiallogeneic pregnancy by reducing expression of CD8, a molecule required for functional activation of CTL.

Gestation stage-dependent intrauterine trophoblast cell invasion in the rat and mouse: novel endocrine phenotype and regulation

Trophoblast cell invasion into the uterine wall is characteristic of hemochorial placentation. In this report, we examine trophoblast cell invasion in the rat and mouse, the endocrine phenotype of invasive trophoblast cells, and aspects of the regulation of trophoblast cell invasion. In the rat, trophoblast cells exhibit extensive interstitial and endovascular invasion. Trophoblast cells penetrate through the decidua and well into the metrial gland, where they form intimate associations with the vasculature. Trophoblast cell invasion in the mouse is primarily interstitial and is restricted to the mesometrial decidua. Both interstitial and endovascular rat trophoblast cells synthesize a unique set of prolactin (PRL)-like hormones/cytokines, PRL-like protein-A (PLP-A), PLP-L, and PLP-M. Invading mouse trophoblast cells also possess endocrine activities, including the expression of PLP-M and PLP-N. The trafficking of natural killer (NK) cells and trophoblast cells within the mesometrial uterus is reciprocal in both the rat and mouse. As NK cells disappear from the mesometrial compartment, a subpopulation of trophoblast cells exit the chorioallantoic placenta and enter the decidua. Furthermore, the onset of interstitial trophoblast cell invasion is accelerated in mice with a genetic deficiency of NK cells, Tgε26 mice, implicating a possible regulatory role of NK cells in trophoblast cell invasion. Additionally, the NK cell product, interferon-γ (IFNγ), inhibits trophoblast cell outgrowth, and trophoblast cell invasion is accelerated in mice with a genetic deficiency in the IFNγ or the IFNγ receptor. In summary, trophoblast cells invade the uterine wall during the last week of gestation in the rat and mouse and possess a unique endocrine phenotype, and factors present in the uterine mesometrial compartment modulate their invasive behavior.

Telomerase activity in human trophoblast

Summary Recent reports have suggested that many proliferating normal cells express telomerase activity, although telomerase activity was previously considered to be specific for cancer cells. This fact draws attention to the investigation of telomerase activity in trophoblast cells which have characteristics of proliferation, differentiation and invasion. Sub-populations of trophoblast cells were separated by a modification of Kliman's method and magnetic cell sorting with antibodies of integrins. Telomerase activity was assayed semi-quantitatively using fluorescence-based TRAP. Cytological detection of telomerase activity was also examined by the newly developed in situ TRAP procedure and the expression of human TERT was detected by RT-PCR. Telomerase activity was detected in 42% of normal placental tissues, and demonstrated decreased activity with gestational age. Telomerase activities was closely correlated with expression of TERT. In sub-populations of CT separated from placenta from early and term gestation, telomerase activity was predominantly detected in columnar CT from early placenta. Fluorescence signals after in situ TRAP were observed in the trophoblastic cell layers in early placenta as well as isolated CT, but not in term placenta. These results suggest that telomerase activity correlates with the regulation of trophoblastic cell proliferation and in situ TRAP is useful for the detection of telomerase positive cells.

Platelet-endothelial cell adhesion molecule-1 is expressed by a subpopulation of human trophoblasts: a possible mechanism for trophoblast-endothelial interaction during haemochorial placentation.

The terminal event in the establishment of the haemochorial placenta in the human is the invasion of trophoblasts into the maternal vessels, a process in which trophoblasts interact directly with the vascular endothelium and degrade the vascular basement membrane and the tunica elastica of the vessels. To further understand this heterotypic cellular interaction, we investigated the expression by human trophoblasts of the vascular cell adhesion molecule platelet-endothelial cell adhesion molecule-1 (PECAM-1) as a possible mediator of the adhesive interaction between trophoblasts and endothelium. In vitro, human trophoblasts were found to express PECAM-1 mRNA and protein. Indirect immunofluorescence indicated a diffuse staining pattern, which was most intense in a subpopulation of trophoblast cells. Co-incubation of trophoblasts with endothelial cells showed interaction between these two cell types with strong expression of PECAM-1 at points of trophoblast-endothelial cell contact, suggesting that this cell adhesion molecule participates in this heterotypic cell interaction. Immunohistochemical localization of PECAM-1 in chorionic villi and first trimester implantation sites showed that, in vivo, only extravillous interstitial and endovascular trophoblasts were positive. In first trimester placentae, villous trophoblast and extravillous trophoblast in other locations than around or within the decidual vessels did not express this molecule. In term placentae, villous trophoblast did not express these adhesion molecules except for two specimens examined. This study demonstrates that PECAM-1 is expressed by a subset of human trophoblasts in vitro and in vivo. Its tissue localization suggests that PECAM-1 is important in mediating the adhesive interaction between trophoblasts and maternal vascular endothelium during the process of haemochorial placentation. Regulation of PECAM-1 expression by human trophoblasts may play a critical role in normal and abnormal vascular invasion during implantation and placentation.

HLA class I expression on the materno-fetal interface.

The conditions that permit the genetically dissimilar (haplo-non-identical) human fetus to evade rejection by its mothers immune system have been the subject of intense interest for several years. As the placental cells, which are in contact with maternal blood or tissue, are devoid of HLA class II antigens, the interest has focused on the expression of HLA class I molecules. Recent findings on the constitutive, transcriptional, and translational expression of HLA class I molecules on anatomically and morphologically different subpopulations of trophoblast cells will form the basis of this article. The expression of HLA class I molecules in the trophoblast cells, forming the materno-fetal junctional zone is inhomogeneous. It differs depending on the differentiation and location of trophoblast cells within the placenta and furthermore on the stage of gestation. On the transcriptional level HLA-A, -B, -C, -E, and -G could be detected on individual trophoblast populations, whereas only HLA-C and HLA-G seem to be translated to protein. The expression of HLA class I antigens by trophoblast cells is not simply suppressed. Instead, less polymorphic HLA-G and HLA-C antigens are carefully selected from the class I multigene family. This gives rise to the assumption that these two HLA class I molecules play an important role in the maintenance of pregnancy.

HLA expression at the maternal-fetal interface.

Pregancy in the human presents an “immunological paradox,” because of the unexpected willingness of mothers to accept genetically disparate tissues. The fact that the fetus can develop unharmed for nine months shows that protective mechanisms must exist to permit its survival. The conditions that permit the genetically dissimilar human fetus to evade rejection by its mother's immune system have been the subject of intense interest for several decades. As the placental cells, which are in contact with maternal blood or tissue, are devoid of HLA class II antigens, interest has focused on the expression of HLA class molecules. Recent developments in the constitutive, transcriptional, and translational expression of HLA class I molecules on anatomically and morphologically different subpopulations of trophoblast cells will form the basis of this short review.

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.

Interferon-gamma rescues HLA class Ia cell surface expression in term villous trophoblast cells by inducing synthesis of TAP proteins.

Human placental trophoblast cells that constitute the materno-fetal interface during pregnancy escape maternal alloimmune attack. The different trophoblast cell subpopulations have developed efficient regulatory mechanisms to prevent expression of beta2-microglobulin-associated HLA class Ia molecules at their cell surface. We previously reported the presence of HLA class Ia messages in villous cytotrophoblast cells and in the syncytiotrophoblast differentiated in vitro purified from term placenta. In this study, we found that these transcripts are translated in heavy chain proteins that are endoglycosidase H sensitive and thus retained in the endoplasmic reticulum or cis-Golgi. Moreover, these class Ia heavy chains can be co-immunoprecipitated with the chaperone protein calnexin resident in the endoplasmic reticulum. When these trophoblast cells are treated with interferon (IFN)-gamma, part of the class Ia heavy chains become endoglycosidase H resistant, demonstrating that they have left the endoplasmic reticulum. Furthermore, after such a treatment, these heavy chains are detectable at the cell surface of these trophoblast cells, as assessed by two-color flow cytometry analysis and immunoprecipitation of cell surface biotinylated proteins using the W6/32 anti-HLA class I monoclonal antibody (mAb). IFN-gamma treatment induces a significant enhancement of the transcription of transporters associated with antigen processing (TAP1 and TAP2) rather than an increase of HLA class I or beta2-microglobulin messages. Finally, we demonstrate that an anti-TAP1 mAb co-immunoprecipitates TAP1 proteins and HLA class Ia heavy chains in these IFN-gamma-treated trophoblast cells. Thus, the constitutive absence of HLA class Ia cell surface expression in term villous cytotrophoblast and syncytiotrophoblast is likely to be due to a lack of transporter proteins that participate in the proper assembly of these molecules in the endoplasmic reticulum. Such a defect can be modified upon IFN-gamma treatment.

HLA-G and pregnancy.

Recent studies of the nonclassical HLA-G class I gene provide insight into its function(s) during pregnancy. The HLA-G gene can be transcribed in different isoforms resulting from alternative splicings and encoding membrane-bound and soluble proteins. These different mRNA species have been found in the various trophoblast cell subpopulations that constitute the maternofetal interface in the human placenta. The raising of antibodies to HLA-G has introduced new tools to determine in which types of trophoblast cells and in which other tissues these transcriptional isoforms are translated in functional proteins. The HLA-G gene exhibits a certain amount of polymorphism, the exon three that encodes the alpha 2 external domain showing the most extensive nucleotide variability. It remains to be determined whether the homozygosity of some HLA-G alleles constitutes a real disadvantage in terms of pregnancy or resistance to specific pathogens. Regarding the potential antigen-presenting function(s) of HLA-G, two isoforms are capable of binding an identical set of nonamer peptides derived from a variety of intracellular proteins. The ligand motif contains three anchor residues and is similar to that of classical HLA class I molecules. Experiments are being performed to identify the recognizing cells and to determine whether HLA-G induces a cytolytic (including anti-viral) T-cell response or in some other way represses natural killer-cell functions.

HLA-G and pregnancy

Recent studies of the nonclassical HLA-G class I gene provide insight into its function(s) during pregnancy. The HLA-G gene can be transcribed in different isoforms resulting from alternative splicings and encoding membrane-bound and soluble proteins. These different mRNA species have been found in the various trophoblast cell subpopulations that constitute the materno‐ fetal interface in the human placenta. The raising of antibodies to HLA-G has introduced new tools to determine in which types of trophoblast cells and in which other tissues these transcriptional isoforms are translated in functional proteins. The HLA-G gene exhibits a certain amount of polymorphism, the exon three that encodes the α2 external domain showing the most extensive nucleotide variability. It remains to be determined whether the homozygosity of some HLA-G alleles constitutes a real disadvantage in terms of pregnancy or resistance to specific pathogens. Regarding the potential antigen-presenting function(s) of HLA-G, two isoforms are capable of binding an identical set of nonamer peptides derived from a variety of intracellular proteins. The ligand motif contains three anchor residues and is similar to that of classical HLA class I molecules. Experiments are being performed to identify the recognizing cells and to determine whether HLA-G induces a cytolytic (including anti-viral) T-cell response or in some other way represses natural killer-cell functions.

Placental expression of HLA class I genes.

This article presents an overview of the more recent data dealing with the constitutive, transcriptional, and translational expression of classical class Ia and nonclassical HLA-E and -G class Ib products in the different trophoblast cell subpopulations that constitute the maternofetal interface during human pregnancy. Of particular interest is the expression of alternatively spliced HLA-G transcriptional isoforms that may be translated in membrane-bound or soluble protein products. Molecular regulatory mechanisms that may control the differential expression of class Ia and class Ib molecules, according to the cell types, state of differentiation, and stages of gestation are also examined. They may operate at the levels of transcription, translation and/or transport of proteins to the cell surface. Functional significance of the absence of detectable cell surface expression of class Ia molecules in all trophoblast cell subpopulations, and of the presence of membrane-bound HLA-G products in extravillous cytotrophoblast cells is finally questioned.

Metalloproteinase activity has a role in equine chorionic girdle cell invasion.

Chorionic girdle cells are a highly invasive subpopulation of trophoblast cells of the equine conceptus. By Day 35 (Day 0 = day of ovulation), cells of the chorionic girdle adhere to the uterine epithelium and begin to invade the endometrial wall. Invasive cells must attach to extracellular matrix proteins, secrete proteinases capable of degrading matrix, and migrate through the degraded matrix; invasion is largely dependent on the proteinase activity of the cells. The objective, therefore, was to develop an in vitro system to examine the mechanisms of equine chorionic girdle cell invasion through extracellular matrix. Day 34 chorionic girdle cell preparations were cultured on Matrigel Invasion Chambers. The cultured invasive cells were binucleate with prominent nucleoli and were often highly vacuolated, consistent with in vivo cup cell morphology. In addition, the cultured cells produced eCG. Additional Day 34 chorionic girdle cell preparations were cultured on Matrigel Invasion Chambers with or without proteinase inhibitors (aprotinin, bestatin, 1,10-phenanthroline) to determine the proteinase activity associated with girdle cell invasion. Only the metalloproteinase inhibitor, 1,10-phenanthroline, inhibited chorionic girdle cell invasion through Matrigel. Chorionic girdle cell supernatants were characterized by zymography, and the proteinases produced by these cells were confirmed to be metalloproteinases at approximate molecular masses of 72 and 95 kDa. The results indicated that equine chorionic girdle cells have matrix-degrading capabilities through metalloproteinase activity. Similar metalloproteinase activity has been reported to be necessary for mouse and human trophoblast invasion, suggesting a similar mechanism of implantation.

Expression of the interferon-γ receptor gene in mouse placentas is related to stage of gestation and is restricted to specific subpopulations of trophoblast cells

In order to evaluate the potential of placental cells to bind the multifunctional cytokine, interferon-gamma (IFN-gamma), tissues collected from pregnant Swiss mice were analysed for IFN-gamma receptor (IFN-gamma R) mRNA and protein. Northern blot hybridization studies indicated that the relative abundance of IFN-gamma R mRNA increased as gestation progressed to term. Analysis by in situ hybridization revealed that trophoblast cells first contained high steady state levels of IFN-gamma R mRNA at g.d. 12. At g.d. 12 and 14, transcription was restricted to cells in the spongiotrophoblast region and nests of similar cells in the labyrinthine region. These cells also contained immunoreactive IFN-gamma R protein. By g.d. 18, IFN-gamma R mRNA was clearly detectable in large spongiotrophoblast cells and labyrinthine trophoblast. IFN-gamma R mRNA was low to absent in giant trophoblast cells at all stages of gestation. Specific mRNA was present in parietal and visceral yolk sac cells by g.d. 14. Thus, expression of the IFN-gamma R gene in mouse placental cells is influenced by stage of gestation, cell lineage and state of differentiation. Whether or not these cells respond vigorously to IFN-gamma with induction of antiviral proteins, increased MHC class I antigens and growth modulation may therefore be determined by their expression of specific receptors for this pluripotent cytokine.