Zone Trophoblast Research Articles

Histological Analysis of Trophoblast Cells in the Mouse Placental Labyrinth Zone.

The mouse is a common animal species used for translational studies. In reproductive studies, this animal is typically preferred over other models as the rodent placenta shows similarities to the human but has a relatively short gestational period. In mice, the transport of oxygen and nutrients between mother and fetus occurs in a restricted area of the placenta called the labyrinth zone. Here, we provide a detailed protocol to study labyrinth zone trophoblast proliferation and syncytial trophoblast identification using paraffin-embedded histological sections of the mouse placenta and immunohistochemistry. By describing step by step how to collect the mouse placenta and process and analyze the labyrinth zone, we hope to help other scientists understand the contribution of changes in placental transport function in their experimental model and therefore advance our understanding of mechanisms underlying pregnancy complications.

Maternal Iron Deficiency Alters Trophoblast Differentiation and Placental Development in Rat Pregnancy.

Iron deficiency, which occurs when iron demands chronically exceed intake, is prevalent in pregnant women. Iron deficiency during pregnancy poses major risks for the baby, including fetal growth restriction and long-term health complications. The placenta serves as the interface between a pregnant mother and her baby, and it ensures adequate nutrient provisions for the fetus. Thus, maternal iron deficiency may impact fetal growth and development by altering placental function. We used a rat model of diet-induced iron deficiency to investigate changes in placental growth and development. Pregnant Sprague-Dawley rats were fed either a low-iron or iron-replete diet starting 2 weeks before mating. Compared with controls, both maternal and fetal hemoglobin were reduced in dams fed low-iron diets. Iron deficiency decreased fetal liver and body weight, but not brain, heart, or kidney weight. Placental weight was increased in iron deficiency, due primarily to expansion of the placental junctional zone. The stimulatory effect of iron deficiency on junctional zone development was recapitulated in vitro, as exposure of rat trophoblast stem cells to the iron chelator deferoxamine increased differentiation toward junctional zone trophoblast subtypes. Gene expression analysis revealed 464 transcripts changed at least 1.5-fold (P < 0.05) in placentas from iron-deficient dams, including altered expression of genes associated with oxygen transport and lipoprotein metabolism. Expression of genes associated with iron homeostasis was unchanged despite differences in levels of their encoded proteins. Our findings reveal robust changes in placentation during maternal iron deficiency, which could contribute to the increased risk of fetal distress in these pregnancies.

Fetal growth restriction is a host specific response to infection with an impaired spiral artery remodeling-inducing strain of Porphyromonas gingivalis

Porphyromonas gingivalis is a periodontal pathogen implicated in a range of pregnancy disorders that involve impaired spiral artery remodeling (ISAR) with or without fetal growth restriction (FGR). Using a rodent periodontitis model, we assessed the ability of P. gingivalis to produce ISAR and FGR in Sprague Dawley (SD) and Wistar (WIS) rats. Both infected SD and WIS rats developed ISAR, but only WIS rats developed FGR despite both rat strains having equivalent microbial loads within the placenta. Neither maternal systemic inflammation nor placental (fetal) inflammation was a feature of FGR in WIS rats. Unique to infected WIS rats, was loss of trophoblast cell density within the junctional zone of the placenta that was not present in SD tissues. In addition, infected WIS rats had a higher proportion of junctional zone trophoblast cells positive for cytoplasmic high temperature requirement A1 (Htra1), a marker of cellular oxidative stress. Our results show a novel phenomenon present in P. gingivalis-induced FGR, with relevance to human disease since dysregulation of placental Htra1 and placental oxidative stress are features of preeclamptic placentas and preeclampsia with FGR.

Alcohol exposure impairs trophoblast survival and alters subtype-specific gene expression in vitro

Maternal alcohol consumption is common prior to pregnancy recognition and in the rat results in altered placental development and fetal growth restriction. To assess the effect of ethanol (EtOH) exposure on the differentiation of trophoblast stem (TS) cells, mouse TS lines were differentiated in vitro for 6 days in 0%, 0.2% or 1% EtOH. This reduced both trophoblast survival and expression of labyrinth and junctional zone trophoblast subtype-specific genes. This suggests that fetal growth restriction and altered placental development associated with maternal alcohol consumption in the periconceptional period could be mediated in part by direct effects on trophoblast development.

Invasion and genome reproduction of the trophoblast cells of placenta junctional zone in the field vole, Microtus rossiaemeridionalis

In the field vole Microtus rossiaemeridionalis, like in other rodents, invasive secondary giant trophoblast cells (SGTC) form a continuous layer at the foeto-maternal interface in the beginning of placentation. However, in the field vole, at midgestation, clusters of junctional zone (JZ) trophoblast non-giant cells interrupt SGTC layer and progressively replace SGTC at the border of decidua basalis. As a result, 'border' cells form a continuous stratum of cytokeratin-positive glycogen-rich cells at the foeto-maternal interface. SGTC plunge into JZ and line the lacunae with maternal blood. SGTC are bound by their highly cytokeratin-positive sprouts forming a framework that holds other trophoblast cell populations. According to DNA cytophotometry, the 'border' cells show the highest ploidy among the JZ cells (up to 46% of 8c cells). Thus, in M. rossiaemeridionalis the role of barrier between semiallogenic foetal and maternal tissues is shifted from the highly endopolyploid (32c-1024c) SGTC to the specific subpopulation of glycogen-rich non-giant (2c-16c) 'border' trophoblast cells that, however, exceed the ploidy of the deeply located and/or proliferative JZ trophoblast cells.

Novel Spatiotemporal Glycome Changes in the Murine Placenta During Placentation Based on BS‐I Lectin Binding Patterns

Although spatiotemporal changes of the glycome (full set of glycans, otherwise known as saccharides or carbohydrates) during placenta formation (placentation) are functionally and clinically important, they are poorly defined. Here, we elucidated novel aspects of the glycome during mouse placentation, from embryonic day 6.5 (E6.5) to E12.5, by investigating the largely unexplored binding distribution of lectin I from Bandeiraea simplicifolia (BS-I lectin), a glycan-binding protein that recognizes the DGalNAc and DGal glycans found at the terminal ends of specific oligosaccharides attached to lipids or proteins. We show that BS-I lectin binding marks all trophoblast cells during early placentation (E7.5 and E8.5 stages), continues in labyrinthine and junctional zone trophoblast but is lost from parietal trophoblast giant cells by E10.5/E11.5 (definitive placenta stage) and is lost from all trophoblast types, but marks the fetal capillary endothelium of the labyrinth, by E12.5. In the decidua basalis (the maternal part of the placenta), BS-I lectin positivity mainly marks the decidual stroma cells of the venous sinusoid area (E7.5 and E8.5 stages) and the entire decidua basalis by E10.5, as well as the osteopontin-positive subset of uterine natural killer (uNK) cells from E7.5 onwards. This work provides the first comprehensive description of the hitherto ill-defined spatiotemporal binding distribution of BS-I lectin in the fetal and maternal placenta between E6.5 and E12.5, thereby contributing to glycome elucidation during placentation. It also establishes BS-I lectin positivity as a novel pan-trophoblast marker during early placentation and as a new marker for mature uNK cells from E7.5 onwards.

New Insights for Ets2 Function in Trophoblast using Lentivirus-Mediated Gene Knockdown in Trophoblast Stem Cells

Mouse trophoblast stem (TS) cells represent a unique in vitro system that provides an unlimited supply of TS cells for the study of trophoblast differentiation and TS cell self-renewal. Although the mouse transcription factor Ets2 is required for TS cell self-renewal, its role in this and in TS cell differentiation has not been explored fully, partly due to the early lethality of Ets2 null mice. To address this, we developed a novel lentivirus-based system that resulted in efficient Ets2 knockdown in the overwhelming majority of TS cells. This system enables functional studies in TS cells, especially for genes required for TS cell self-renewal because TS cell derivation using gene-knockout embryos for such genes depends on TS cell self-renewal. Using morphological/morphometric criteria and gene expression analysis, we show that the requirement for Ets2 in self-renewal of TS cells cultured in ‘stem cell medium’ (SCM) involves maintenance of the expression of genes that inhibit TS cell differentiation in SCM, such as Cdx2 and Esrrb, and preservation of the undifferentiated TS cell morphology. During TS cell differentiation caused by Cdx2/ Esrrb downregulation, due to either Ets2 knockdown in SCM or culture in differentiation medium (DM), Ets2 is also required for the promotion of trophoblast giant cell (TGC) and junctional zone trophoblast (JZT) differentiation. This TGC differentiation involves Ets2-dependent expression of Hand1, a gene required for the differentiation of all TGC types. This study uncovers new roles for Ets2 in TS cell self-renewal and differentiation and demonstrates the usefulness of this lentivirus system for gene function studies in TS cells.

Csf2 Null Mutation Alters Placental Gene Expression and Trophoblast Glycogen Cell and Giant Cell Abundance in Mice1

Genetic deficiency in granulocyte-macrophage colony-stimulating factor (CSF2, GM-CSF) results in altered placental structure in mice. To investigate the mechanism of action of CSF2 in placental morphogenesis, the placental gene expression and cell composition were examined in Csf2 null mutant and wild-type mice. Microarray and quantitative RT-PCR analyses on Embryonic Day (E) 13 placentae revealed that the Csf2 null mutation caused altered expression of 17 genes not previously known to be associated with placental development, including Mid1, Cd24a, Tnfrsf11b, and Wdfy1. Genes controlling trophoblast differentiation (Ascl2, Tcfeb, Itgav, and Socs3) were also differentially expressed. The CSF2 ligand and the CSF2 receptor alpha subunit were predominantly synthesized in the placental junctional zone. Altered placental structure in Csf2 null mice at E15 was characterized by an expanded junctional zone and by increased Cx31(+) glycogen cells and cyclin-dependent kinase inhibitor 1C (CDKN1C(+), P57(Kip2+)) giant cells, accompanied by elevated junctional zone transcription of genes controlling spongiotrophoblast and giant cell differentiation and secretory function (Ascl2, Hand1, Prl3d1, and Prl2c2). Granzyme genes implicated in tissue remodeling and potentially in trophoblast invasion (Gzmc, Gzme, and Gzmf) were downregulated in the junctional zone of Csf2 null mutant placentae. These data demonstrate aberrant placental gene expression in Csf2 null mutant mice that is associated with altered differentiation and/or functional maturation of junctional zone trophoblast lineages, glycogen cells, and giant cells. We conclude that CSF2 is a regulator of trophoblast differentiation and placental development, which potentially influences the functional capacity of the placenta to support optimal fetal growth in pregnancy.

Placental Expression of Peroxisome Proliferator-Activated Receptors in Rat Pregnancy and the Effect of Increased Glucocorticoid Exposure1

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors. Recent gene deletion studies indicate that PPARG and PPARD play critical roles in rodent development, including effects on placental vascularization. In this study we investigated the expression of the PPAR isoforms and their heterodimeric partner, RXRA, in the two functionally and morphologically distinct zones of the rat placenta during normal gestation and after glucocorticoid-induced fetal and placental growth restriction. Real-time reverse transcription-polymerase chain reaction and immunohistochemical analysis demonstrated markedly higher expression of Ppara, Pparg, and Rxra mRNA in labyrinth zone trophoblast as compared with basal zone near term. There was also a marked increase in Pparg (65%, P < 0.05) and Ppara (91%, P < 0.05) mRNA specifically in the labyrinth zone over the final third of pregnancy. In contrast, expression of Ppard mRNA fell (P < 0.001) in both placental zones over the same period. Maternal dexamethasone treatment (1 mug/ml in drinking water; Days 13-22, term = 23 days) reduced placental (44%) and fetal (31%) weights and resulted in a fall in Pparg (37%, P < 0.05) mRNA expression specifically in the labyrinth zone at Day 22. Placental expression of Ppara, Ppard, and Rxra was unaffected by dexamethasone treatment. These data suggest that PPARG:RXRA heterodimers play important roles in labyrinth zone growth late in pregnancy, possibly supporting vascular development. Moreover, glucocorticoid inhibition of placental growth appears to be mediated, in part, via a labyrinth-zone-specific suppression of PPARG.

Phagocytosis as a potential mechanism for microbial defense of mouse placental trophoblast cells.

Trophoblast giant cells are active phagocytes during implantation and post-implantation. Phagocytosis decreases during placental maturation as the phagocytic function of nutrition is gradually replaced by the direct uptake of nutrients by the labyrinth zone trophoblast. We hypothesize that, after placental maturation, trophoblast cells maintain phagocytic functions for purposes other than nutrition. This study employs histological techniques to examine the ability of trophoblast cells to phagocytose microorganisms (yeast or bacteria)--in vivo in females receiving thioglycolate to activate macrophages and in vitro in the presence of phagocytic promoters such as interferon-gamma and complement component C3. Placental trophoblast cells from the second half of gestation show basal phagocytosis that can be dramatically up-regulated by these promoters when microorganisms are inoculated into pregnant animals or introduced into culture systems. Stimulated trophoblast cells phagocytosed organisms more rapidly and in greater numbers than non-stimulated trophoblast exposed to the same numbers of organisms. Taken together, our results indicate that trophoblast cells do not lose their ability to phagocytose during the placentation process, which may imply that trophoblast cells participate in embryonic and fetal innate immune defense through elimination of microorganisms present at the maternal-fetal interface.

Migratory trophoblast cells express a newly identified member of the prolactin gene family.

Rodents possess an expanded prolactin (PRL) family of genes. These genes encode for a family of structurally related hormones/cytokines that are expressed most prominently in the anterior pituitary, uterus and placenta. In this study, we have identified a new member of the rat PRL family through a search of the National Center for Biotechnology Information expressed sequence database. The cDNA was sequenced and its corresponding mRNA characterized. On the basis of existing nomenclature, the rat cDNA was termed PRL-like protein-N (PLP-N). PLP-N has structural features indicative of its inclusion in the PRL family and is most closely related to PRL-like protein-F (PLP-F) and proliferin related protein (PLF-RP). A survey of PLP-N mRNA expression by Northern analysis indicated that PLP-N showed extensive expression in the metrial gland and minimal expression in the chorioallantoic placenta or other tIssues. Expression of PLP-N mRNA was restricted to migratory trophoblast cells. Junctional zone trophoblast cells isolated from day 13 of gestation placenta differentiated in vitro and exhibited a capacity for PLP-N expression. In summary, we have discovered a new member of the PRL family that is prominently expressed in migratory trophoblast cells residing in the metrial gland.

Lectin binding characteristics of mouse placental cells.

The lectin binding characteristics of mouse placental cells were examined. Wax embedded tissue sections of placentae from d 14 pregnant mice were stained with 26 lectins, with a wide range of sugar specificities. Cell cultures prepared from d 14 mouse placentae and cultured for 24 h were stained with 7 of the lectins to determine if they could be used as markers for the different trophoblast cells in culture. In tissue sections all placental cell populations bound lectin but no lectin bound specifically to any single trophoblast population. All the lectins which bound to layer 1 cytotrophoblast lining the maternal blood spaces of the labyrinthine placenta also bound to the fetal endothelium of the labyrinthine placenta. Binding of lectin appeared strongest on the adluminal membrane of these cell populations suggesting a role for the carbohydrate moieties in nutrient transfer. Few lectins bound to junctional zone trophoblast. Overall, the binding of lectin to cultured cells did not correlate exactly with lectin binding to the cell populations in tissue sections. The value of lectins as markers for placental cells in culture was therefore found to be limited. Our findings indicate that carbohydrate expression by at least some placental cells may vary in culture from that expressed by the cells in vivo with obvious concerns for the validity of functional in vitro studies.

Inactivated X-chromosome as an interphase chromosome marker for evaluation of mechanisms of genome multiplication in the rat and rabbit placental trophoblast cells

One of the main peculiarities of differentiation of giant trophoblast cells (GTCs) that are first to establish a close interconnection with the allogenic maternal tissues is their passing to the endoreduplication, i.e., a modified cell cycle excluding mitoses and leading to genome multiplication. The inactivated X-chromosome (Barr body) has been used as a chromosome marker that allows the chromosome behavior in the interphasic nucleus to be traced in the course of genome multiplication. Rat and rabbit GTCs of different ploidy levels (16–25c) have been found to contain a single Barr body. The simultaneous cytophotometric measurement of the DNA content in the Barr body and in the entire nucleus have shown that an increase of the DNA content in the single Barr body parallels elevation of the total nuclear DNA amount in the course of genome multiplication from 16 to 256c. The ratio of these values was constant at all ploidy degrees. These data confirm the polytenic nature of the GTCs. In contrast, nuclei of the junctional zone trophoblast cells (JTCs), which can divide mitotically up to the octaploid level, contained 1–4 Barr bodies, the percentage of cells with 2–4 Barr bodies increasing with the ploidy level. Thus, in the JTCs the genome multiplication seems to be accompanied by a chromosome number increase that is characteristic of “restitutional” mitoses. Therefore, the most invasive GTCs pass to the endoreduplication cycle, which leads to polyteny, whereas JTCs that do not contact with maternal tissues can undergo restitution.

Isolation and characterization of rat trophoblast cells.

Previous immunohistochemical studies of the rat placenta using specific alloantisera and/or monoclonal antibodies showed that the basal zone trophoblasts stained for Pa and Aa class I major histocompatibility complex (MHC) antigens and for the human SP1-related antigen. In an effort to isolate the basal zone trophoblast cells from the rat placenta, we used these markers to assess the degree of purification of the cells separated by density gradient centrifugation using either Ficoll-Hypaque or Percoll as the gradient medium. The cells were put either on the top or at the bottom of discontinuous density gradients in the range of 1.005-1.10 g/ml. The cell separation profiles for the two media were different. With Percoll, most of the trophoblast cells (80-95%) were collected at the density gradients 1.04/1.06 and 1.06/1.08, whereas with Ficoll-Hypaque, these gradients separated only a small fraction (4-23%) of the trophoblast cells, and most of them pelleted at the bottom of the tube. The trophoblast cells separated by Ficoll-Hypaque, however, showed fewer contaminant cells than those separated by the Percoll gradients.

Ontogeny of Expression of Pa and RT1.Aa Antigens on Rat Placenta and on Fetal Tissues

The unique pregnancy-associated (Pa) antigen, which is a class I antigen encoded by the major histocompatibility complex (MHC), elicits a nondestructive maternal antibody response. By contrast, the class I transplantation antigen RT1.Aa elicits a destructive antibody response in tissue transplantation but not during pregnancy. With the use of the avidin-biotin complex (ABC) immunohistochemical method, the Pa and RT1.Aa antigens were localized on the basophilic and giant cells of the basal zone trophoblast, the endovascular trophoblast and decidual interstitial trophoblast, and the chorioallantoic membrane but not on the labyrinthine zone trophoblast as early as the 12th day of gestation. These two antigens were also expressed on the epidermis, hair follicles, spleen, thymic medulla, bronchial epithelium, intestinal epithelium, the hepatic Kupffer cells, endocardium, endothelium of blood vessels, renal tubular cells and glomeruli, and renal pelvis and ureter of fetal and adult rat tissues. Absorption studies with placental tissue confirmed the presence of these two antigens in the rat placenta, and antibody-blocking studies confirmed their unique specificities.

The rat placenta expresses paternal class I major histocompatibility antigens

The expression of paternally inherited class I MHC antigens on the placental trophoblast of the rat has been investigated using a mouse anti-rat monoclonal antibody (MN4-91-6) in an indirect immunoperoxidase labelling assay on cryostat sections. Strong specific staining was obtained on the spongy zone trophoblast of the mature placenta from DA ♂ (RT1 a) × PVG ♀ (RT1 c) matings. In marked contrast, no staining was observed on the labyrinthine trophoblast nor on the trophoblastic giant cells at any stage of gestation from 8 to 19 days post-coitum. None of the trophoblastic cell populations at any stage of gestation were reactive with an anti-class II monoclonal antibody. Class I positive endovascular cytotrophoblast cells were present in the maternal arterial sinusoids of the decidua. These findings imply that maternal immunoregulatory mechanisms must be essential for the survival of the placenta and fetus.

The immunohistochemical localization of pregnancy-specific β1-glycoprotein in postimplantation rat trophoblast

Pregnancy-specific beta 1-glycoprotein (SP1) was identified by peroxidase-antiperoxidase (PAP) immunohistochemistry in the placenta of inbred strains of rat between 10 and 21 days of gestation. SP1 was located predominantly in basal zone trophoblast and in intravascular trophoblast of decidual vessels, but it was absent from labyrinthine trophoblast. No perivascular cells were identified by SP1 staining, but occasional clusters of interstitial trophoblast stained for SP1. The results suggest that basal and labyrinthine trophoblast are functionally different.

Inhibition of progesterone synthesis in placenta by administration of dexamethasone to pregnant rats

Glucocorticoid receptors have been detected in placenta from several species, including the rat, although the biological function of corticoids is unknown in placenta from the latter species. The present experiments examined the effect of glucocorticoid treatment on placental progesterone biosynthesis from endogenous precursors by incubated basal zone trophoblast and labyrinthine zone of placentas from adrenalectomized-ovariectomized rats at the end of pregnancy. It was found that a higher proportion of synthesized progesterone was retained in the tissue than that released into the incubation medium. Treatment of rats on the 17th–18th day of pregnancy with 10μg/ml of dexamethasone in the drinking saline for 3 days, produced a significant inhibition of progesterone detected in tissue and medium of incubated placental zones. In vitro addition of dexamethasone (10 −4 M) was also effective in reducing progesterone in the placental zone studied (LZ). Serum progesterone of intact rats was in the range of rats near parturition (approx 25ng/ml) and dropped to almost undetectable levels in rats with adrenalectomy and ovariectomy, with or without dexamethasone treatment, suggesting that in late pregnancy the rat placenta does not contribute significantly to circulating levels of progesterone. This glucocorticoid effect could not be extended to estrogens, as we, in accord with the work of other groups, failed to detect estrogen synthesis in rat placenta. It is suggested that a function for glucocorticoid receptors in rat placenta may be the inhibition of local progesterone production.

The placenta as a glucocorticoid target tissue: exchange assay of (3H)-dexamethasone binding and effect of steroids on receptor content.

Binding of (3H)-dexamethasone was studied in high speed supernatant (HSS) of basal zone trophoblast and labyrinthine zone of rat placenta using an exchange assay. The system showed the following characteristics: (1) maximum binding was attained after 120 min of incubation; (2) molybdate was required in the medium to measure binding sites at 20 degrees C and equilibrium conditions; (3) exchange was near 90-100% between ligand and corticosterone added during a preincubation; (4) low capacity (190-250 fmoles/mg protein), high affinity (Kd 10(-8) M) binders were determined by saturation analysis; (5) competition with other steroids in vitro revealed that in basal zone trophoblast, only dexamethasone displaced the ligand, whereas in labyrinthine zone corticosterone, progesterone and testosterone competed to a smaller degree than dexamethasone. We have also studied the effect of in vivo treatment of pregnant rats for four days with dexamethasone, corticosterone, estradiol, progesterone or testosterone, on (3H)-dexamethasone binding. Of all tested compounds, only dexamethasone treatment (which was suspended 24 h before the experiment to prevent receptor occupancy by exogenous hormone) significantly decreased binding of the tritiated hormone in HSS of labyrinthine zone and basal zone trophoblast. These results suggest that dexamethasone regulates its own receptor in placenta. The physiological aspect of this phenomenon requires elucidation of the role of glucocorticoids on placental function in the rat.

Binding of [3H]dexamethasone by rat placenta.

[3H]Dexamethasone binding was measured in the basal zone trophoblast (BZT) and the labyrinthine zone (LZ) of placenta from Wistar rats on days 19–22 of pregnancy. Cytosol from both zones was incubated with [3H]dexamethasone for 4 h at 0–4 C, and high affinity, low capacity sites were measured by Scatchard plot analysis in BZT and LZ, tissues which demonstrated similar concentrations of sites and affinity constants. Specific binding was decreased by omitting mercaptoethanol from the incubation buffer, by adding N-ethylmaleimide, or by increasing the temperature to 16 or 37 C. Competition studies revealed that the rank order of inhibition of [3H]dexamethasone binding by nonradioactive steroids was dexamethasone » progesterone = corticosterone » testosterone = 17β-estradiol. Dexamethasone-binding sites could be distinguished from progestinbinding sites by competition analysis as well as by the absence of 17α,21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione ([3H]-R5020) binding in LZ, whereas the corticoid was ...