Labyrinthine Trophoblast Research Articles

Placental Expression of 2,3 Bisphosphoglycerate Mutase in IGF-II Knock out Mouse: Correlation of Circulating Maternal 2,3 Bisphosphoglycerate and Fetal Growth

Bisphosphoglycerate mutase (BPGM) catalyses the formation of 2,3 bisphosphoglycerate (BPG) a ligand of haemoglobin. BPG facilitates liberation of oxygen from haemoglobin at low oxygen tension enabling efficient delivery of oxygen to tissues. We describe expression of BPGM in mouse labyrinthine trophoblasts, located at the maternal–placental interface. Expression is lower in placentae of igf2 +/− knockout mice, a widely used model of growth restriction, compared to wild type placentae. Circulating maternal BPG increased throughout gestation but this increase was less in wt mothers carrying igf2 +/− pups than in those carrying exclusively wt pups. This reduction was observed well before term and may contribute to the low birth weight of igf2 +/− pups. Strikingly, we also measured reductions of fetal and placental weight in wt littermates of igf2 +/− pups compared to pups developing in an exclusively wt environment. These data suggest that placental expression of BPGM can influence maternal BPG concentrations and supports a hypothesis under which BPG synthesized in the placenta may act on maternal haemoglobin to enhance delivery of oxygen to the developing fetus.

Etoposide Induces TRP53-Dependent Apoptosis and TRP53-Independent Cell Cycle Arrest in Trophoblasts of the Developing Mouse Placenta1

Abnormal regulation of placental apoptosis and proliferation has been implicated in placental disorders. Recently, several DNA-damaging agents were reported to induce excessive apoptosis and reduce cell proliferation in the placenta; however, the molecular pathways of these toxic effects on the placenta are unclear. The aim of the present study was to determine the involvement of TRP53, a tumor suppressor that mediates cellular responses to DNA damage, in the induction of apoptosis and cell cycle arrest in the developing placenta. For this purpose, we treated pregnant mice on Day 12 of gestation with 10 mg/kg of etoposide and 5-Gy gamma irradiation, potent inducers of DNA damage. We found an increase in the number of trophoblastic apoptoses 8 and 24 h after etoposide injection and 6 and 24 h after irradiation in the placental labyrinth zone. The number of mitoses and DNA syntheses in trophoblasts decreased after treatment. The accumulation and phosphorylation of TRP53 protein were detected 8 and 6 h after etoposide injection and irradiation, respectively. In Trp53-deficient placentas, the induction of etoposide-induced trophoblastic apoptosis is abrogated, while the reduction of proliferation occurred similarly as in wild-type placentas. CDC2A, a regulator of G2/M progression, was inactivated by phosphorylation after etoposide injection and irradiation, suggesting that the cell cycle was arrested at the G2/M border by treatment. Our study demonstrated that etoposide injection induced TRP53-dependent apoptosis and TRP53-independent cell cycle arrest in labyrinthine trophoblasts, providing insights into the molecular pathway of placental disorders.

Overexpression of thioredoxin-1 reduces oxidative stress in the placenta of transgenic mice and promotes fetal growth via glucose metabolism.

Oxidative stress occurs where there is an imbalance between the production and scavenging of free radicals. Pregnancy per se is a state of oxidative stress due to the increased metabolic activity of placental mitochondria and reduced scavenging ability of antioxidant systems. Overproduction of reactive oxygen species may be associated with impaired fetal growth. However, the physiological influence of antioxidant systems on fetal growth is not well understood. In this study we assessed the effects of antioxidant systems on fetal growth using human thioredoxin (hTRX)-1 overexpressing transgenic (Tg) mice. Tg or C57BL/6 [wild-type (WT)] male mice were mated with WT female mice, and dams were killed to obtain the fetuses and placentas on gestational d 15. Tg fetuses were significantly heavier than WT fetuses, whereas placental weight did not differ significantly between the two groups. Immunohistochemically, hTRX-1 was localized to the nuclei of labyrinthine trophoblasts in Tg mice. In addition, placental expression of 8-hydroxy-2'-deoxyguanosine, which reflects DNA damage caused by oxidative stress, was reduced in Tg mice compared with WT mice. Placental expression of glucose transporter-1 mRNA and protein was significantly higher in Tg mice than WT mice, whereas no significant differences were observed for glucose transporter-3, IGF, and IGF-binding protein mRNA expression. These results suggest that placental and/or systemic antioxidant systems can influence fetal growth. In particular, increased hTRX-1 activity and the resulting modified placental redox state may play an important role in fetal growth by increasing the availability of glucose.

Regulation of natural-killer cell cytotoxicity and enhancement of complement factors in the spontaneously aborted mouse placenta

To evaluate the cytotoxicity of uterine natural killer (NK) cells and identify major biological factors fluctuating in the spontaneously aborted placenta. Spontaneously aborted placentae were examined by using histology, molecular biology, and DNA microarray analysis. Laboratories and animal center at a medical college. C57BL/6 mice. None. Up-regulation of the inhibitory system of uterine NK cells and enhancement of complement factors in aborted placenta. In uteri on day 14 of pregnancy, 9.69% +/- 11.6% (+/-SD) implantation sites spontaneously aborted. Labyrinthine trophoblasts at abortion sites had nuclei that were positive for DNA fragment detection, as well as apoptotic morphology. Many uterine NK cells were present at abortion sites, but cytotoxic factors such as perforin, granzyme B, and Fas-Fas ligand were expressed at very low levels. In contrast, the expression level of Ly49 NK-cell receptors, which mediate an inhibitory signal for cytotoxicity, was enhanced at abortion sites. Deoxyribonucleic acid microarray analysis showed that adipsin, an activating enzyme for complement component C3, was strongly enhanced in aborted placenta, and positive reactions for adipsin and C3 were confirmed by immunohistochemistry. In the process of spontaneous abortion, the cytotoxicity of uterine NK cells is inhibited, and the innate immune system through adipsin and complement C3 appears to be influential.

Effects of maternal nutrient restriction on placental morphology and insulin-like growth factor system expressions

Uteroplacental insufficiency is one of the major causes of fetal growth restriction (FGR) in developed countries. The pathophysiology is likely the result of fetal undernutrition due to the impairment of nutrient and substrate transfer from the mother to the fetus. Fetal undernutrition due to maternal undernutrition is not a major cause of FGR in developed countries compared to developing countries. However, FGR can be reproducibly created in experimental animals by maternal undernutrition, and is commonly utilized to study the pathophysiology of fetal undernutrition in the fetus and the placenta. The insulin-like growth factor (IGF) system is the principal growth factor involved in regulating normal fetal and placental growth and its aberrant expression is associated with the development of FGR. We hypothesized that placenta undergoes adaptive changes in pregnancies with maternal undernutrition, the effects of which are mediated by the IGF system. 
 Methods: A mouse model of FGR was generated in CD-1 mice using maternal total caloric nutrient restriction. At 6.5 days of pregnancy, mice were randomly assigned to the control group (ad libitum diet) or global nutrient restriction (GNR) diet group (70% of predetermined food intake of each gestational day). FGR can be reproducibly induced in the fetuses without effect in litter size. Placentae were collected at 18.5 days and were fixed in 4% paraformaldehyde. Five mm tissue sections were prepared by standard techniques and were stained with hematoxylin & eosin for morphometry, PAS for glycogen staining, and immunohistochemistry against IGF-I and –II, IGFBP-1, -2 and –3 using specific antisera for cell-specific expression of the IGF system. The morphology of the placentae was analyzed using a computerized image analysis system. 
 Results: Placentae were significantly smaller in FGR mice compared to controls (94±4 mg SEM vs 72±3 mg SEM). Preliminary results demonstrate changes in FGR mouse placentae tended to be a result of a reduction in spongiotrophoblast layer (p = 0.066, one-tailed t-test) while the labyrinthine trophoblast layer was maintained. Morphometric analysis of the labyrinth demonstrated an increase in the diameter of both maternal and fetal vessels with a reduction in the thickness of the interchorial layer. The IGF-I and –II, IGFBP-1, IGFBP-2 and IGFBP-3 immunoreactivity were all reduced in FGR placentae compared to the controls. 
 Conclusion: These findings suggest that maternal nutrient restriction induces changes in the placenta that may potentially lead to improved nutrient and substrate transfer from mother to fetus, in an attempt to maintain fetal viability. However, these compensatory changes are not sufficient to maintain growth. These changes may result from the alteration of expression of the IGF system genes.

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is essential for the integrity of basement membranes in the developing placental labyrinth

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a membrane-associated Kunitz-type serine protease inhibitor that regulates cell surface and extracellular serine proteases involved in tissue remodeling and tumorigenesis, such as HGFA, matriptase, prostasin and hepsin. We generated HAI-1-deficient mice, which died in utero due to placental defects. The HAI-1 −/− placental labyrinth exhibited a complete failure of vascularization and a compact morphology of the trophoblast layer. Immunofluorescent staining of collagen IV and laminin and electron microscopy analysis revealed that this aberrant labyrinth architecture was associated with disrupted basement membranes located at the interface of chorionic trophoblasts and allantoic mesoderm. Unlike the placental labyrinth, basement membranes and vasculogenesis were normal in embryo and yolk sac. Therefore, basement membrane defects appear to be the underlying cause for the greatly impaired vascularization and trophoblast branching in HAI-1 −/− placentas. In wild-type placentas, the expression of matriptase and prostasin co-localized with their physiological inhibitor HAI-1 to the labyrinthine trophoblast cells in proximity to basement membranes. In HAI-1 −/− placentas, both the localization and expression of the two proteases remained unchanged, implying uncontrolled proteolytic activities of the two enzymes. Our study demonstrates the important role of HAI-1 in maintaining the integrity of basement membrane most likely by regulating extracellular proteolytic activities during placental development.

Expression Profiling of Mouse Placental Lactogen II and Its Correlative Genes Using a cDNA Microarray Analysis in the Developmental Mouse Placenta

The placenta is a highly differentiated organ essential for embryonic growth and development. In order to search for key molecules that are associated with mouse placental lactogen II (mPL-II) gene expression, we applied mouse cDNA microarray analysis to RNAs extracted from placentae on days 10, 12, 14, 16 and 18 of pregnancy. Changes in gene expression were categorized between days 10 and 12, 12 and 14, 14 and 16 and 16 and 18 of pregnancy. After microarray analysis, which had a minimum detectable fold change for differential expression of 2, we selected 10 genes, Apoa2, Apoc2, Ceacam14, Creg1, Fmo1, Igf2, Slc2a1, Spink3, Spi1-1 and Tpbpa, exhibiting a expression pattern similar to the mPL-II gene. Furthermore, we performed real-time PCR analysis and in situ hybridization (ISH) to find correlative expression genes for the mPL-II gene. From these results, we identified a resemblance in gene expression between mPL-II and Igf2 and selected these genes for performance of double-fluorescence immunohistochemical staining. We colocalized these proteins in labyrinthine trophoblast cells. These results strongly suggest that the expression of mPL-II and Igf2 is highly related to placental development in mice. This large-scale identification of genes regulated during placentogenesis assists in further elucidation of the molecular basis of extraembryonic development and function.

Origin and characteristics of glycogen cells in the developing murine placenta

The junctional zone (Jz) of the mouse placenta consists of two main trophoblast populations, spongiotrophoblasts and glycogen cells (GCs), but the development and function of both cell types are unknown. We conducted a quantitative analysis of GC size, number, and invasion of cells into the decidua across gestation. Furthermore, we identified markers of GC function to investigate their possible roles in the placenta. While the spongiotrophoblast cell volume doubles, and cell number increases steadily from E12.5 to E16.5, there is a remarkable 80-fold increase in GC numbers. This finding is followed by a notable decrease by E18.5. Surprisingly, the accumulation of GCs in the decidua did not fully account for the decrease in GC number in the Jz, suggesting loss of GCs from the placenta. Glucagons were detected on GCs, suggesting a steady glucose release throughout gestation. Connexin31 staining was shown to be specific for GCs. GC migration and invasion may be facilitated by temporally regulated expression of matrix metalloproteinase 9 and the imprinted gene product, Decorin. Expression of the clearance receptor for type II insulin-like growth factor (IGF-II), IGF2R, in a short developmental window before E16.5 may be associated with regulating the growth effects of IGF-II from glycogen cells and/or labyrinthine trophoblast on the expansion of the Jz. Thus stereology and immunohistochemistry have provided useful insights into Jz development and function of the glycogen cells.

879. Effects of Continuous Expression of Activated Protein C (APC) in Novel Murine Thrombosis Models

Top of pageAbstract APC mediates anticoagulant functions and signals cellular responses that are anti-inflammatory in nature. To date, APC is the only drug efficacious in human with severe sepsis. Because of its potential role in human diseases other than sepsis, we sought to exploit APC's effects in models of thrombosis. However, assessment of APC's in vivo role has been compromised by its short half-life (15 min). To overcome this limitation, we used adeno-associated viral (AAV) vectors encoding the PC zymogen or APC for liver-specific expression. Initial experiments in mice demonstrated that APC, but not PC, expression (50|[ndash]|400 ng/ml) is effective in preventing in vivo thrombus formation at both macro and microcirculation, with minimal risk of bleeding. Here we described a novel murine model for thrombophilia characterized by fetal loss and spontaneous adult thrombosis. We crossed mice with FVL mutation that renders the protein resistant to APC's proteolytic activity with mice expressing elevated levels of FIX (tFIX). Mice that achieved adulthood developed normally until 7 month of age when 10% of FVL(+/|[minus]|)/tFIX and 5% of FVL(|[minus]|/|[minus]|)/tFIX mice showed spontaneous thrombosis, whereas no other age-matched mice (n=200) developed such complications. Breeding of mice FVL(+/|[minus]|) with mice tFIX generated more than 400 newborns of all genotypes but none of FVL(+/+)/tFIX genotype. Interestingly, the number of reabsorbed embryos increased from 13% (E9.5) to 33% (E16.5, p<0.02), and reached 54% when the mother was tFIX. All FVL(+/+)/tFIX embryos exhibited extensive bruises and hemorrhagic areas which colocalize with marked fibrin deposition, findings consistent with disseminated intravascular coagulation (DIC). Placental analysis (n=20) of FVL(+/+)/tFIX embryos harvested at E12.5 or later demonstrated extensive abnormalities, such as excessive fibrin deposition and necrotic cells (17%) in the labyrinthine trophoblast. These findings were not found in hundreds of embryos of other genotypes or in placental tissues. The maternal genotype also influenced the number of newborns per litter that was significantly lower when the mother carried tFIX compared with father tFIX (4.8 vs. 8.3, P<0.02). Pregnant mice (n=12) received injection of a thrombin inhibitor (hirudin) at doses ranging from 0.3 to 0.6 mg/kg/day throughout the gestation. This strategy resulted in reduced placental fibrin deposition and necrotic areas (6% vs. 17% of untreated mice, p<0.01), but did not rescue FVL(+/+)/tFIX mice. Moreover, the rate of reabsorbed embryos and the litter size were not affected by the anticoagulant. We next injected mice with AAV-APC into female mice FVL(+/|[minus]|)tFIX (n=6) and crossed them with male FVL(+/|[minus]|). The results indicate that APC expression is associated with low numbers of reabsorbed embryos (20%) compared to those gestations without AAV-APC (54%, p<0.02). These data suggest that rescue of embryonic lethality of prothrombotic phenotype models affecting the protein C pathway requires both antithrombotic and anti-inflammatory effects that unlikely can be provide by anticoagulant drugs other than APC.

Transforming Growth Factor-βInduces Differentiation of the Labyrinthine Trophoblast Stem Cell Line SM10

The mammalian placenta consists of different trophoblast cell types that assist in the variety of functions required for the maintenance of pregnancy. In rodents, labyrinthine trophoblasts of the placenta are especially important, because they are capable of differentiating into fused labyrinthine cells, which form the feto-maternal exchange surface. Even though the molecular signals triggering labyrinthine trophoblast differentiation are poorly understood, transforming growth factor-beta (TGF-beta) has been shown to be present in the placental environment and alter trophoblast development. In this study, we investigated the effects of TGF-beta on the differentiation of the labyrinthine trophoblast stem cell lines SM10 and HRP-1. RT-PCR analyses demonstrated that while the molecular expression of labyrinthine-specific lineage markers (Esx1, Tfeb, and Tec) was maintained in TGF-beta-treated SM10 and HRP-1 cells, TGF-beta induced the down-regulation of trophoblast stem cell markers Id2 and Cdx2. In contrast, TGF-beta induced the expression of a marker of differentiated labyrinthine trophoblasts, Gcm1, only in the SM10 cell line. Furthermore, we demonstrated an increased glucose uptake in the TGF-beta-treated SM10 cells, indicative of functional differentiation. Finally, cell fusion in TGF-beta-treated SM10 and HRP-1 cells was investigated by western blotting analysis of placental alkaline phosphatase and cadherin-11 and by microscopic analyses of cell morphology using green fluorescent protein (GFP) and rhodamine phalloidin staining. The western blotting and morphological analyses indicate TGF-beta-induced cell fusion and morphological differentiation in the SM10 cell line. The SM10 cell line will provide a new and unique model for detailed analysis of TGF-beta-induced molecular events associated with labyrinthine trophoblast differentiation and function.

Hypoxia-Inducible Factors 1α and 2α Regulate Trophoblast Differentiation

ABSTRACT Placental development initially occurs in a low-oxygen (O 2 ) or hypoxic environment. In this report we show that two hypoxia-inducible factors (HIFs), HIF1α and HIF2α, are essential for determining murine placental cell fates. HIF is a heterodimer composed of HIFα and HIFβ (ARNT) subunits. Placentas from Arnt − / − and Hif1α − / − Hif2α −/− embryos exhibit defective placental vascularization and aberrant cell fate adoption. HIF regulation of Mash2 promotes spongiotrophoblast differentiation, a prerequisite for trophoblast giant cell differentiation. In the absence of Arnt or Hifα , trophoblast stem cells fail to generate these cell types and become labyrinthine trophoblasts instead. Therefore, HIF mediates placental morphogenesis, angiogenesis, and cell fate decisions, demonstrating that O 2 tension is a critical regulator of trophoblast lineage determination. This novel genetic approach provides new insights into the role of O 2 tension in the development of life-threatening pregnancy-related diseases such as preeclampsia.

Ultrastructural changes in the interhaemal membrane and junctional zone of the murine chorioallantoic placenta across gestation

The mouse is an extremely useful experimental model for the study of human disease owing to the ease of genetic and physiological manipulation. A more detailed knowledge of murine placental development will, we hope, increase our understanding of the pathogenesis of placentally related complications of human pregnancy. The murine placenta consists of two main fetally derived compartments: the labyrinthine zone and the junctional zone. Exchange in the labyrinthine zone takes place across an interhaemal membrane comprising an outer layer of cytotrophoblast cells and two inner layers of syncytial trophoblast. The cytotrophoblast layer thins as gestation advances, and in addition becomes highly perforated after embryonic day (E)12.5. Furthermore, as gestation advances cytotrophoblast nuclear volume and DNA content increase, suggesting the formation of labyrinthine trophoblast giant cells. The syncytial layers become increasingly microvillous, enlarging the surface area for exchange. Separate basement membranes support the syncytium and the fetal capillary endothelium throughout gestation, although these appear to fuse where the capillaries are closely approximated to the trophoblast. The junctional zone consists of two principal trophoblast cell types, spongiotrophoblasts and invasive glycogen cells, yet the functions of each remain elusive. Spongiotrophoblasts vary in their appearance even when not fully differentiated, but a striking feature is the extensive endoplasmic reticulum of the more mature cells. Early glycogen cells are distinguished by the presence of electron-dense glycogen granules, and large amounts of surrounding extracellular matrix. Later the accumulations of glycogen granules occupy almost all the cytoplasm and there are few organelles. This is the first study to use both scanning and transmission electron microscopy in an ultrastructural description of murine placental development and is complementary to contemporary genetic investigations.

Co-Inheritance of FV Leiden and High Levels of FIX Results in Novel Models for Thrombophilia and Spontaneous Fetal Loss.

High levels of FIX is emerging as a risk factor for spontaneous venous thrombosis, affecting ~20% of unselect patients and increasing rates of recurrent thromboembolic events. We have generated mice expressing supraphysiological levels of human FIX (up to 400% of normal) but these animals did not develop thrombosis. Therefore, we decided to test whether an additional risk for thrombosis would trigger a prothrombotic phenotype in these mice. Because the FVL mutation is also present in 20% of thrombophilia subjects, we chose FVL mice as model. Breedings included both parents being heterozygous FVL(+/−) but only one was transgenic for FIX (tFIX: 4–8μg/ml plus the endogenous murine FIX). All mice were on C57Bl/6 background. More than 200 newborns were obtained but surprisingly no animal of FVL(+/+)/tFIX genotype was identified. When pregnancies were interrupted at embryonic age of E9.5 to E16.5, no deviation from the expected/observed embryos genotypes was observed. However, the number of reabsorbed embryos increased significantly from 13% (E9.5) to 33% (E.16.5), p<0.02. All FVL(+/+)/tFIX embryos exhibited extensive bruises and hemorrhagic areas which colocalize with markedly fibrin deposition, findings consistent with disseminated intravascular coagulation. Placental analysis (n=18) of FVL(+/+)/tFIX embryos harvested at E12.5 or later demonstrated abnormalities such as excessive fibrin deposition and innumerous apoptotic cells, determined by TUNEL assay, mainly in the labyrinthine trophoblast zone. The maternal genotype clearly influenced the pregnancy outcome since the number of newborns per litter was 2-fold lower when the mother carried tFIX compared with father tFIX (average 4.3 vs. 8.3, respectively; p<0.02). No mice of other genotypes analyzed (n=112) presented similar abnormalities neither in embryos nor in placental tissue. Mice that achieve adulthood developed normally. However, FVL(+/−)tFIX mice (>7 months of age) presented high rate of thrombotic episodes (3/40; 7.5%) followed by 5.2% FVL(−/ −)/tFIX mice, whereas no other age-matched mice (n=74) developed such complication. Monitoring for coagulation activation by serial aPTT, PT, thrombin-antithrombin (TAT) and D-Dimers demonstrated genotype-dependent procoagulant activity. In FVL(+/−)/tFIX or FVL(−/ −)/tFIX mice, levels of TAT (70ng/ml, n=28/group) and D-dimers (230ng/ml, n=17/group) were ~ 2-fold higher (P<0.04), when compared to mice (TAT: 30ng/ml and D-Dimer: 100ng/ml) of other genotypes. Furthermore, FVL(+/−)/tFIX mice presented the highest fibrin deposition determined by Western blot analysis of tissues harvested, followed by FVL(−/ −)/tFIX mice when compared to the other animals (P<0.05). To exclude the possibility of abnormal interaction of human (h) FIX with murine antithrombin (AT), we compared the rate of inactivation of hFIXa by murine or human AT using purified proteins. The results showed that murine AT efficiently inactivated hFIXa in a similar fashion of that determined for human AT. Moreover, early work showed that hFIX efficiently corrected the phenotype of FIX-deficient mice. Therefore, these data revealed that gene-dosage dependent interaction of FVL and high levels of FIX in mice increase rates of spontaneous fetal loss and adulthood thrombosis. These models provide opportunities to better understand and to test therapeutics for thrombophilia-related complications due to common risk factors identified in humans.

Identification of Committed Placental Stem Cell Lines for Studies of Differentiation

Trophoblasts provide a model to investigate fundamental mechanisms of stem cell differentiation, but the availability of trophoblast stem cell lines is limited. Here we report the development of an RT-PCR-based lineage-specific profile as a method to identify the lineages of placental trophoblast cells routinely and specifically. This profiling method was used to analyze the mouse SM10 and rat HRP-1 cell lines, isolated from a region of the placental labyrinth, but of previously unidentified lineage. Using this profile, the expression of trophoblast stem cell markers was detected in the SM10 and HRP-1 cells. In contrast, no expression of a marker of differentiated labyrinthine trophoblast was detected. Additionally, both cell lines expressed labyrinthine trophoblast-specific genes and did not express lineage-specific markers of spongiotrophoblasts or trophoblast giant cells. Our results suggest that SM10 and HRP-1 cell lines are trophoblast stem cell-like cell lines that can be maintained in undifferentiated but committed state in cell culture. These cell lines express labyrinthine-specific genes and are committed to differentiate solely into functional labyrinthine trophoblasts. Our profiling method provides a new technique to identify stem cells and their lineage-specific differentiation. This method additionally indicates that SM10 and HRP-1 cell lines provide new systems for future studies of stem cell differentiation, allowing investigation of basic mechanisms of differentiation, which may provide insights into the biophysics of development of a specialized system. This method should also prove to be useful for identification of other stem cell lines and examination of lineage-specific commitment.

The Expression of Insulin-like Growth Factor and Insulin-like Growth Factor Binding Protein mRNAs in Mouse Placenta

Insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) are paracrine regulators of tissue growth and development, and are expressed at the sites of biological action. To study the role of the IGFs and IGFBPs in mouse placental development, we determined the temporal and spatial expression patterns of the mRNAs at embryonic days 10.5 to 18.5 by in situ hybridization. IGF-II mRNA was expressed strongly in mesoderm and fetal blood vessels of early placenta and in labyrinthine trophoblast of later placenta. In the junctional zone, IGF-II mRNA was expressed first in spongiotrophoblasts, later strongly in glycogen cells and variably in giant cells. IGFBP-2 mRNA was expressed weakly in spongiotrophoblasts and glycogen cells. IGFBP-2, -5 and -6 mRNAs were detected in the stroma of the metrial gland. Myometrium expressed IGFBP-2 mRNA strongly, IGFBP-6 mRNA moderately and IGFBP-5 mRNA weakly. The endothelium of maternal blood vessels in decidua expressed IGFBP-3 and -5 mRNAs, and some deeper vessels expressed IGFBP-4 mRNA. In the yolk sac, IGF-II mRNA was expressed in endoderm and mesoderm, whereas IGFBP-1, -2 and -4 mRNAs were expressed only in endoderm, and IGFBP-4 mRNA in mesoderm. Strong expression of IGF-II mRNA in glycogen cells suggests a role in the autocrine/paracrine regulation of invasion. Similar to rat and guinea pig, but in contrast to man and primates, IGFBP mRNAs, except IGFBP-4, were not expressed in mouse decidua. However, IGFBP-3, -4 and -5 mRNAs were expressed in endothelium of maternal blood vessels, and IGFBP-2 and -6 mRNAs in myometrium, where IGFBPs may play a critical role in regulating trophoblast invasion. These findings suggest possible biological roles of the peptides at the feto-maternal interface.

Peroxisome Proliferator-Activated Receptor γ Controls Muc1 Transcription in Trophoblasts

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is essential for placental development. Here, we show that the mucin gene Muc1 is a PPARgamma target, whose expression is lost in PPARgamma null placentas. During differentiation of trophoblast stem cells, PPARgamma is strongly induced, and Muc1 expression is upregulated by the PPARgamma agonist rosiglitazone. Muc1 promoter is activated strongly and specifically by liganded PPARgamma but not PPARalpha or PPARdelta. A PPAR binding site (DR1) in the proximal Muc1 promoter acts as a basal silencer in the absence of PPARgamma, and its cooperation with a composite upstream enhancer element is both necessary and sufficient for PPARgamma-dependent induction of Muc1. In the placenta, MUC1 protein is localized exclusively to the apical surface of the labyrinthine trophoblast around maternal blood sinuses, resembling its luminal localization on secretory epithelia. Last, variably penetrant maternal blood sinus dilation in Muc1-deficient placentas suggests that Muc1 regulation by PPARgamma contributes to normal placental development but also that the essential functions of PPARgamma in the organ are mediated by other targets.

The Product of the Imprinted Gene IPL Marks Human Villous Cytotrophoblast and is Lost in Complete Hydatidiform Mole

The IPL/TSSC3 gene is expressed nearly exclusively from the maternal allele, and its protein product acts to limit placental growth in mice. This protein specifically marks Type II trophoblast in the labyrinthine layer of the mouse placenta. To investigate mouse–human homologies, we carried out immunohistochemistry with antibodies against human IPL. There was strong expression of IPL in villous cytotrophoblast of the human placenta, contrasting with complete lack of expression in syncytiotrophoblast. Staining for IPL was weak in cells of the villous mesenchyme and extravillous trophoblast, including the cytotrophoblast columns in the basal plate and the intervillous trophoblast islands. The IPL and p57 KIP2 /CDKN1C genes are closely linked and coordinately imprinted, and immunostaining showed that their protein products are co-expressed in villous cytotrophoblast. However, other cell types, including extravillous cytotrophoblast and cells in various non-placental tissues, expressed p57 KIP2, but not IPL. IPL protein was absent in both of two cases of androgenetic complete hydatidiform mole examined by immunostaining, and IPL mRNA was absent in an additional three cases of this neoplasm examined by northern blotting. In the mouse, Ipl-expressing cells disappear at mid- to late-gestation when placental growth ceases, but persistent IPL mRNA and protein expression was observed throughout human gestation, correlating with the continuous growth of the human placenta. These findings highlight dosage regulation of human IPL by imprinting and, more generally, suggest homology between Type II labyrinthine trophoblast in the mouse and villous cytotrophoblast in humans, both of which are proliferative stem cell-like compartments.

The IGF-II-deficient placenta: aspects of its function

Gene ablation in mice has provided compelling evidence of the role that insulin-like growth factors (IGFs) and their receptors play in fetal and placental development. The recent deletion of the P0 transcript encoding IGF-II specifically within the labyrinthine trophoblast, resulted in placental and fetal growth restriction. This restriction was associated with reduced placental permeability and increased system A amino acid transporter activity. Whilst this report provides important new data on the role of placental IGF-II, they might not recapitulate the changes that occur in human placental insufficiency leading to intrauterine growth restriction.

Placental-specific IGF-II is a major modulator of placental and fetal growth.

Imprinted genes in mammals are expressed from only one of the parental chromosomes, and are crucial for placental development and fetal growth. The insulin-like growth factor II gene (Igf2) is paternally expressed in the fetus and placenta. Here we show that deletion from the Igf2 gene of a transcript (P0) specifically expressed in the labyrinthine trophoblast of the placenta leads to reduced growth of the placenta, followed several days later by fetal growth restriction. The fetal to placental weight ratio is thus increased in the absence of the P0 transcript. We show that passive permeability for nutrients of the mutant placenta is decreased, but that secondary active placental amino acid transport is initially upregulated, compensating for the decrease in passive permeability. Later the compensation fails and fetal growth restriction ensues. Our study provides experimental evidence for imprinted gene action in the placenta that directly controls the supply of maternal nutrients to the fetus, and supports the genetic conflict theory of imprinting. We propose that the Igf2 gene, and perhaps other imprinted genes, control both the placental supply of, and the genetic demand for, maternal nutrients to the mammalian fetus.

Differential contributions of AF-1 and AF-2 activities to the developmental functions of RXRα

We have engineered a mouse mutation that specifically deletes most of the RXR alpha N-terminal A/B region, which includes the activation function AF-1 and several phosphorylation sites. The homozygous mutants (RXR alpha af1(o)), as well as compound mutants that further lack RXR beta and RXR gamma, are viable and display a subset of the abnormalities previously described in RXR alpha-null mutants. In contrast, RXR alpha af1(o)/RAR(-/-)(alpha, beta or gamma) compound mutants die in utero and exhibit a large array of malformations that nearly recapitulate the full spectrum of the defects that characterize the fetal vitamin A-deficiency (VAD) syndrome. Altogether, these observations indicate that the RXR alpha AF-1 region A/B is functionally important, although less so than the ligand-dependent activation function AF-2, for efficiently transducing the retinoid signal through RAR/RXR alpha heterodimers during embryonic development. Moreover, it has a unique role in retinoic acid-dependent involution of the interdigital mesenchyme. During early placentogenesis, both the AF-1 and AF-2 activities of RXR alpha, beta and gamma appear to be dispensable, suggesting that RXRs act as silent heterodimeric partners in this process. However, AF-2 of RXR alpha, but not AF-1, is required for differentiation of labyrinthine trophoblast cells, a late step in the formation of the placental barrier.