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Anatomical organization of the mature placenta and the maternal spiral arteries. The arrows highlight the orientation of tissue sectioning for sagittal and transverse sections. Sagittal tissue sections are used for the routine analysis of mor- phology and marker gene expression. Serial transverse sections are used for detailed assessment of endovascular trophoblast invasion.
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Placental development is a dynamic and complex process and much of our current understanding of the underlying molecular processes comes from analysis of targeted gene mutations in mice. There are more than 50 strains of mutant mice that have placental defects, and it has become widely appreciated that placental defects should be suspected in cases...
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... this may be a simple oversight, because it has been only 20_Natale_273_294_F 8/29/05, 11:21 AMrecently that the normal patterns of invasion have been described and methods developed to identify the two distinct invading cell populations (9). Sagittal sections can be used to generally survey the extent of invasion, but serial cross sections ( Fig. 5) are needed in order to study the course of invasion of the endovascular trophoblast giant cells because they traffic along spiral shaped arteries that come in and out of the planes of sagittal sections. The endovascular giant cells are apparent from the early postimplantation period to term and are characterized by positive staining ...
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Local tissue oxygenation profoundly influences placental development. To elucidate the impact of hypoxia on cellular and molecular adaptation in vivo, pregnant mice at embryonic days 7.5-11.5 were exposed to reduced environmental oxygen (6-7% O2) for various periods of time. Hypoxia-inducible factor (HIF)-1alpha mRNA was highly expressed in the pla...
Citations
... Endogenous alkaline phosphatase (AP) staining was used to identify the maternal blood spaces, while isolectin B4 (L5391; Sigma) staining was used to identify the basement membrane of the fetal capillaries (herein referred to as fetal blood space) in the labyrinth layer of the placenta, as previously described [32,33]. ...
... Of the genes analyzed, the marker most elevated on fibronectin was Gcm-1. In the mouse, the Gcm-1-positive SynTII cells of the labyrinth are adjacent to the fibronectincontaining basement membrane of the fetal vessels [10,32]. In the STZ placentae, the SynTII population was reduced, alongside reduced fibronectin. ...
Trophoblast stem (TS) cells were first isolated from the mouse placenta; however, little is known about their maintenance and niche in vivo. TS cells, like other stem cells, have a unique microenvironment in which the extracellular matrix (ECM) is a component. Placental pathology is associated with ECM change. However, how these changes and the individual ECM components impact the maintenance or differentiation of TS cells, has not been established. This study identified which ECM component(s) maintain undifferentiated mTS cells and which alter their differentiation profile. Additionally, the mouse model of streptozotocin (STZ)-induced pancreatic beta-cell ablation and diabetes was used as a model of placental pathology to investigate whether placental ECM influenced the expression of genes associated with different trophoblast sub-types. mTS cells were cultured on individual ECM components and qPCR analysis revealed that laminin promoted the expression of markers associated with undifferentiated TS cells, fibronectin promoted gene expression associated with syncytiotrophoblast (SynT) layer II cells and collagen IV promoted differentiation primarily to junctional zone trophoblast. Female mice administered STZ (blood glucose 300 mg/dL) or control (blood glucose 150 mg/dL) were mated. Placental pathology at embryonic day (E)14.5 was confirmed with reduced fetal blood space area, reduced expression of the pericyte marker SMA, and decreased expression of ECM proteins. mTS cells cultured on ECM isolated from STZ placenta were associated with reduced expression of undifferentiated mTS markers and SynA (SynT layer I), while expression of Tpbpa (junctional zone) was increased, suggesting that analysis of ECM isolated from the placenta may prove a useful tool in understanding trophoblast contribution to placental pathology.
... Ten placentas were analyzed in this study; five of them were wild type and 5 were mutant (COUP-TFI KO). The placentas were separated from the surrounding tissue according to the previously described technique [19]. Subsequently, sagittal central sections of the placentas were used for the analysis [19]. ...
... The placentas were separated from the surrounding tissue according to the previously described technique [19]. Subsequently, sagittal central sections of the placentas were used for the analysis [19]. ...
Background: Chicken Ovalbumin Upstream Promoter-Transcription Factor I (COUP-TFI) is a member of the steroid/thyroid nuclear receptor superfamily. The aim of this study was to investigate whether absence of this gene affects placental development and fetal growth in a COUP-TFI knockout mouse model. Methods: Placentas of COUP-TFI-knockout (COUP-TFI KO) and wild-type (WT) were collected at 18.5 days post-coitum. The expression level of the following genes known to be involved in different key molecular pathways was evaluated: BCL2 Associated X (Bax) and B-cell lymphoma 2 (Bcl-2) (apoptosis), p21, p53 and α subunit of inhibin (INHA) (proliferation and apoptosis), vascular endothelial growth factor A (VEGF-A), placental growth factor (PlGF), hypoxia-inducible factor 1-alpha (HIF1α), Fms related receptor tyrosine kinase 1 (Flt-1), and endoglin (ENG) (angiogenesis). Mouse litter weight at birth was also assessed. Results: RT-qPCR analysis showed increased mRNA expression of VEGF-A and Bax in placental tissue of COUP-TFI KO mice compared to WT mice. We also found a loss in the positive correlation between Bcl-2 and INHA, p21 and ENG, as well as HIF1α and Flt-1 mRNA expression in COUP-TFI mutants. Finally, KO mice were lighter than WT littermates (respectively, the mean weight of COUP-TFI KO mice was 1.3 grams, ± 0.13, compared to 1.6 g, ± 0.14 of WT mice, p < 0.05). Conclusions: Our results show that COUP-TFI deletion is associated with a lower birth weight in mice and increased placental transcript expression of pro-apoptotic Bax and pro-angiogenetic VEGF-A genes.
... A complete assessment of the placenta should include both macroscopic and microscopic examinations. [15][16][17] The placenta consists of readily discernible embryonic and maternal areas. Key embryonic derivatives include a layered placental disc (comprised from deep to superficial, the chorionic plate, labyrinth, and junctional zone) and membranes that envelop the embryo (amnion and yolk sac). ...
The use of the mouse as a model organism is common in translational research. This mouse–human similarity holds true for placental development as well. Proper formation of the placenta is vital for development and survival of the maturing embryo. Placentation involves sequential steps with both embryonic and maternal cell lineages playing important roles. The first step in placental development is formation of the blastocyst wall (approximate embryonic days [E] 3.0-3.5). After implantation (∼E4.5), extraembryonic endoderm progressively lines the inner surface of the blastocyst wall (∼E4.5-5.0), forming the yolk sac that provides histiotrophic support to the embryo; subsequently, formation of the umbilical vessels (∼E8.5) supports transition to the chorioallantoic placenta and hemotrophic nutrition. The fully mature (“definitive”) placenta is established by ∼E12.5. Abnormal placental development often leads to embryonic mortality, with the timing of death depending on when placental insufficiency takes place and which cells are involved. This comprehensive macroscopic and microscopic atlas highlights the key features of normal and abnormal mouse placental development from E4.5 to E18.5. This in-depth overview of a transient (and thus seldom-analyzed) developmental tissue should serve as a useful reference to aid researchers in identifying and describing mouse placental changes in engineered, induced, and spontaneous disease models.
... Alkaline phosphatase (AP) has been shown to be specifically expressed by the trophoblast cells lining the maternal blood spaces and serves as a marker for these cells [19]. We therefore used AP expression to identify TGCs, as described in previous studies [9,19]. ...
... Alkaline phosphatase (AP) has been shown to be specifically expressed by the trophoblast cells lining the maternal blood spaces and serves as a marker for these cells [19]. We therefore used AP expression to identify TGCs, as described in previous studies [9,19]. Paraffin sections of 5 µm thickness were dewaxed, rehydrated, and washed in NT solution (0.15 M NaCl and 0.1 M Tris, pH 7.5) for 20 min at room temperature followed by a second wash with freshly prepared NTMT solution (0.1 M NaCl, 0.1 M Tris, pH 9.5, 0.05 M MgCl 2 , and 0.1% Tween-20) for 10 min at room temperature. ...
... Paraffin sections of 5 µm thickness were dewaxed, rehydrated, and washed in NT solution (0.15 M NaCl and 0.1 M Tris, pH 7.5) for 20 min at room temperature followed by a second wash with freshly prepared NTMT solution (0.1 M NaCl, 0.1 M Tris, pH 9.5, 0.05 M MgCl 2 , and 0.1% Tween-20) for 10 min at room temperature. AP activity was visualized after incubation with a standard chromogenic AP substrate (BCIP/NBT; Promega), counterstaining with Nuclear Fast Red, dehydrating by passing through a graded ethanol series, and mounting after clearing in xylene [9,19]. ...
Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that acts primarily on endothelial cells, but numerous studies suggest that VEGF also acts on non-endothelial cells, including trophoblast cells. Inhibition of VEGF signaling by excess production of the endogenous soluble VEGF receptor sFlt1 in trophoblast cells has been implicated in several pregnancy complications. Our previous studies and other reports have shown that VEGF directly regulates placental vascular development and functions and that excess VEGF production adversely affects placental vascular development. Trophoblast giant cells (TGCs) line the maternal side of the placental vasculature in mice and function like endothelial cells. In this study, we specifically examined the effect of excess VEGF signaling on TGC development associated with defective placental vascular development using two mouse models an endometrial VEGF overexpression model and a placenta-specific sFlt1 knockdown model. Placentas of endometrial VEGF-overexpressing dams at embryonic days (E) 11.5 and 14.5 showed dramatic enlargement of the venous maternal spaces in junctional zones. The size and number of the parietal TGCs that line these venous spaces in the placenta were also significantly increased. Although junctional zone venous blood spaces from control and VEGF-overexpressing dams were not markedly different in size at E17.5, the number and size of P-TGCs were both significantly increased in the placentas from VEGF-overexpressing dams. In sFlt1 knockdown placentas, however, there was a significant increase in the size of the sinusoidal TGC-lined, alkaline phosphatase-positive maternal blood spaces in the labyrinth. These results suggest that VEGF signaling plays an important role in maintaining the homeostasis of the maternal vascular space in the mouse placenta through modulation of TGC development and differentiation, similar to the effect of VEGF on endothelial cells in other vascular beds.
... Furthermore, mTORC1 inhibition in PHT cells decreased the expression of CRELD1, a member of a subfamily of epidermal growth factor-related proteins. This may be relevant for placental angiogenesis because CRELD1 knock out in mice caused placental abnormalities and global vascular insufficiency in the fetus (Natale et al., 2006). Collectively, these data implicate trophoblast mTORC1 in promoting placental angiogenesis. ...
Mechanistic Target of Rapamycin Complex 1 (mTORC1) serves as positive regulator of placental nutrient transport and mitochondrial respiration. The role of mTORC1 signaling in modulating other placental functions is largely unexplored. We used gene array following silencing of raptor to identify genes regulated by mTORC1 in primary human trophoblast (PHT) cells. Seven hundred and thirty-nine genes were differentially expressed; 487 genes were down-regulated and 252 up-regulated. Bioinformatic analyses demonstrated that inhibition of mTORC1 resulted in decreased expression of genes encoding ribosomal proteins in the 60S and 40S ribosome subunits. Furthermore, down-regulated genes were functionally enriched in genes involved in eIF2, sirtuin and mTOR signaling, mitochondrial function, and glutamine and zinc transport. Stress response genes were enriched among up-regulated genes following mTORC1 inhibition. The protein expression of ribosomal proteins RPL26 (RPL26) and Ribosomal Protein S10 (RPS10) was decreased and positively correlated to mTORC1 signaling and System A amino acid transport in human placentas collected from pregnancies complicated by intrauterine growth restriction (IUGR). In conclusion, mTORC1 signaling regulates the expression of trophoblast genes involved in ribosome and protein synthesis, mitochondrial function, lipid metabolism, nutrient transport, and angiogenesis, representing novel links between mTOR signaling and multiple placental functions critical for normal fetal growth and development.
... AP staining was used to assess for differentiation of STGCs. Briefly, sections were deparaffinized and rinsed in PBS and incubated in wash buffer (NTMT: 0.1 M NaCl; 0.1 M tris, pH 9.5; 0.05 M MgCl 2 ; and 0.1% Tween 20, prepared fresh), and AP activity was detected by using BCIP-NBT (bromochloroindolyl phosphate-nitro blue tetrazolium) substrate to form a blue precipitate as previously described (46). The reaction was stopped in PBS, and cells were counterstained in Nuclear Fast Red (Vector Laboratories, catalog no. ...
Cell-cell fusion or syncytialization is fundamental to the reproduction, development, and homeostasis of multicellular organisms. In addition to various cell type–specific fusogenic proteins, cell surface externalization of phosphatidylserine (PS), a universal eat-me signal in apoptotic cells, has been observed in different cell fusion events. Nevertheless, the molecular underpinnings of PS externalization and cellular mechanisms of PS-facilitated cell-cell fusion are unclear. Here, we report that TMEM16F, a Ca ²⁺ -activated phospholipid scramblase (CaPLSase), plays an essential role in placental trophoblast fusion by translocating PS to cell surface independent of apoptosis. The placentas from the TMEM16F knockout mice exhibit deficiency in trophoblast syncytialization and placental development, which lead to perinatal lethality. We thus identified a new biological function of TMEM16F CaPLSase in trophoblast fusion and placental development. Our findings provide insight into understanding cell-cell fusion mechanism of other cell types and on mitigating pregnancy complications such as miscarriage, intrauterine growth restriction, and preeclampsia.
... Determining the size of the different layers during placentation is therefore considered as a first important step when phenotyping new mutant models. Conventionally, these measurements are assessed by standard histological protocols (12), complemented with additional histologic stains to classify cellular tissue composition. Although this technique has proven to be a valuable tool owing to its high discriminative power and accuracy at cellular level, it is subjected to sampling bias. ...
... Indeed, failure to appreciate placental defects has resulted in the misconception of gene function during development. Initial analysis of the murine placenta involves measuring the areas of the placental layers on histological sections (12). However, layered structures such as the placenta are prone to anisotropic and nonuniform deformation during tissue processing, which is, withal, a process that is sample specific (39). ...
Significance
During pregnancy, the placenta functions as surrogate fetal lungs, kidneys, and gut. Proper placental functioning is therefore paramount during embryonic development. Indeed, placental defects are highly prevalent in mouse mutants showing embryonic death. Here we validate a contrast agent that allows development of the intact placenta to be visualized using microfocus computed topography. This technique enables an initial 3D inspection of the overall placental structure and provides the possibility to quantify different compartments in the placenta. Moreover, the nondestructive nature of this contrast agent permits further histological processing of the same sample when defects are observed.
... Sinusoidal trophoblast giant cells that line the maternal blood spaces express endogenous alkaline phosphatase (AP), thus allowing for their identification (Fig. 2d,e) 45 . The total blood space area (maternal and fetal combined), showed no statistical difference, regardless of gestational age or condition ( Fig. 2f-i). ...
... During early placental development, different trophoblast lineages form in two distinct regions, the junctional zone and the labyrinth. Within these regions, distinct trophoblast cell subtypes are present and can be identified morphologically, as well as by in situ hybridization (Fig. S9a,b) [45][46][47] . To determine the effects of prolonged expression of CA-Hif-1α on trophoblast development during pregnancy, we used established trophoblast lineage markers [45][46][47][48][49] . ...
... Within these regions, distinct trophoblast cell subtypes are present and can be identified morphologically, as well as by in situ hybridization (Fig. S9a,b) [45][46][47] . To determine the effects of prolonged expression of CA-Hif-1α on trophoblast development during pregnancy, we used established trophoblast lineage markers [45][46][47][48][49] . An assessment of labyrinth-specific cell types was conducted by examining the expression of Epcam, for labyrinth trophoblast progenitors, as well as Gcm-1 and Syna, for syncytiotrophoblasts (Figs 3 and S10a-d) 50,51 . ...
The placenta is an essential organ that is formed during pregnancy and its proper development is critical for embryonic survival. While several animal models have been shown to exhibit some of the pathological effects present in human preeclampsia, these models often do not represent the physiological aspects that have been identified. Hypoxia-inducible factor 1 alpha (Hif-1α) is a necessary component of the cellular oxygen-sensing machinery and has been implicated as a major regulator of trophoblast differentiation. Elevated levels of Hif-1α in the human placenta have been linked to the development of pregnancy-associated disorders, such as preeclampsia and fetal growth restriction. As oxygen regulation is a critical determinant for placentogenesis, we determined the effects of constitutively active Hif-1α, specifically in trophoblasts, on mouse placental development in vivo. Our research indicates that prolonged expression of trophoblast-specific Hif-1α leads to a significant decrease in fetal birth weight. In addition, we noted significant physiological alterations in placental differentiation that included reduced branching morphogenesis, alterations in maternal and fetal blood spaces, and failure to remodel the maternal spiral arteries. These placental alterations resulted in subsequent maternal hypertension with parturitional resolution and maternal kidney glomeruloendotheliosis with accompanying proteinuria, classic hallmarks of preeclampsia. Our findings identify Hif-1α as a critical molecular mediator of placental development and indicate that prolonged expression of Hif-1α, explicitly in placental trophoblasts causes maternal pathology and establishes a mouse model that significantly recapitulates the physiological and pathophysiological characteristics of preeclampsia with fetal growth restriction.
... Statistical analysis by two-way ANOVA addressed whether there was a difference between the Sham and RUPP group at each of the gestational ages, and whether there was a difference in response between the two gestational ages. The placenta develops to facilitate exchange of nutrients and waste and as such analysis included the fetal capillary network and maternal blood sinusoids within the labyrinth layer (herein referred to as fetal and maternal blood spaces, respectively [16][17][18][19][20]. This assessment included: area of blood spaces relative to total placental area; change in maternal and/or fetal blood space; maternal to fetal blood space ratio and the perimeter to area ratio, as an indicator of nutrient exchange. ...
... A minimum of 5 to 10 dams were dissected at each stage, for each control and experimental group described below. Placentae were fixed in 4% paraformaldehyde overnight, embedded in paraffin and sectioned for analysis by histological staining; immunohistochemistry and in situ hybridization as previously described 18,75,119 . RUPP experiment surgeries were conducted as previously described in the rat 4 but modified to include bilateral ligation of the uterine arteries with surgical thread instead of a surgical clip and no ligation of the abdominal aorta. ...
Abstract This study characterized the effect of the reduced utero-placental perfusion pressure (RUPP) model of placental insufficiency on placental morphology and trophoblast differentiation at mid-late gestation (E14.5). Altered trophoblast proliferation, reduced syncytiotrophoblast gene expression, increased numbers of sinusoidal trophoblast giant cells, decreased Vegfa and decreased pericyte presence in the labyrinth were observed in addition to changes in maternal blood spaces, the fetal capillary network and reduced fetal weight. Further, the junctional zone was characterized by reduced spongiotrophoblast and glycogen trophoblast with increased trophoblast giant cells. Increased Hif-1α and TGF-β-3 in vivo with supporting hypoxia studies in trophoblast stem (TS) cells in vitro, support hypoxia as a contributing factor to the RUPP placenta phenotype. Together, this study identifies altered cell populations within the placenta that may contribute to the phenotype, and thus support the use of RUPP in the mouse as a model of placenta insufficiency. As such, this model in the mouse provides a valuable tool for understanding the phenotypes resulting from genetic manipulation of isolated cell populations to further understand the etiology of placenta insufficiency and fetal growth restriction. Further this study identifies a novel relationship between placental insufficiency and pericyte depletion in the labyrinth layer.
... Placentae were dissected at different stages of gestation from pregnant CD-1 females. Placentae were fixed in 4% paraformaldehyde overnight, embedded in paraffin and sectioned for analysis by histological staining; immunohistochemistry and in situ hybridization as previously described 17,26,43 . RUPP experiment surgeries were conducted as previously described in the rat 32 and the mouse 34 but modified to include bilateral ligation of the uterine arteries with surgical thread instead of a surgical clip. ...
Trophoblast stem (TS) cells in the mouse derive from the polar trophectoderm of the blastocyst and persist through early gestation (to E8.5) to support placental development. Further development and growth is proposed to rely on layer-restricted progenitor cells. Stem cell antigen (Sca) -1 is a member of the Ly6 gene family and a known marker of stem cells in both hematopoietic and non-hematopoietic mouse tissues. Having identified that Sca-1 mRNA was highly expressed in mouse TS cells in culture, we found that it was also expressed in a subset of trophoblast within the chorion and labyrinth layer of the mouse placenta. Isolation and in vitro culture of Sca-1⁺ trophoblast cells from both differentiated TS cell cultures and dissected mouse placentae resulted in proliferating colonies that expressed known markers of TS cells. Furthermore, these cells could be stimulated to differentiate and expressed markers of both junctional zone and labyrinth trophoblast subtypes in a manner comparable to established mouse TS cell lines. Our results suggest that we have identified a subpopulation of TS cell-like cells that persist in the mid- to late- gestation mouse placenta as well as a cell surface protein that can be used to identify and isolate these cells.
