Expression and localization of SWAP-70 in human fetomaternal interface and placenta during tubal pregnancy and normal placentation.

SWAP-70 has been demonstrated as a multiple functional signaling protein involved in formation of membrane ruffling induced by signal cascade of tyrosine kinase growth factor receptors. In the present study, the spatial and temporal expression pattern of SWAP-70 on human fetomaternal interface was investigated using specimens collected from tubal and normal pregnancies by in situ hybridization, immunohistochemistry, and Western blotting. Data showed an intense expression of SWAP-70 in trophoblasts at weeks 3-6 of fallopian implantation and at weeks 6-7 of normal pregnancy. The most intense expression was exhibited by those highly motile and invasive extravillous trophoblasts. From gestational week 8 on, the level of SWAP-70 in trophoblasts decreased significantly, and the signal was restricted in villous cytotrophoblast cells. In the in vitro cultured human trophoblast cell line, B6Tert-1, colocalization of SWAP-70 with F-actin was verified. Data in human placenta were similar to what we recently reported on rhesus monkey fetomaternal interface. Our results suggest that SWAP-70 may be involved in regulating migration and invasion of trophoblast cells during the processes of embryonic implantation and placentation in primates.     

Differential expression of the metastasis suppressor KAI1 in decidual cells and trophoblast giant cells at the feto-maternal interface

Invasion of trophoblasts into maternal uterine tissue is essential for establishing mature feto-maternal circulation. The trophoblast invasion associated with placentation is similar to tumor invasion. In this study, we investigated the role of KAI1, an anti-metastasis factor, at the maternal-fetal interface during placentation. Mouse embryos were obtained from gestational days 5.5 (E5.5) to E13.5. Immunohistochemical analysis revealed that KAI1 was expressed on decidual cells around the track made when a fertilized ovum invaded the endometrium, at days E5.5 and E7.5, and on trophoblast giant cells, along the central maternal artery of the placenta at E9.5. KAI1 in trophoblast giant cells was increased at E11.5, and then decreased at E13.5. Furthermore, KAI1 was upregulated during the forskolinmediated trophoblastic differentiation of BeWo cells. Collectively, these results indicate that KAI1 is differentially expressed in decidual cells and trophoblasts at the maternal-fetal interface, suggesting that KAI1 prevents trophoblast invasion during placentation. [BMB Reports 2013; 46(10): 507-512]     

Dynamic change of Adamalysin 19 (ADAM19) in human placentas and its effects on cell invasion and adhesion in human trophoblastic cells

Human ADAM19 is a recently identified member of the ADAM family. It is highly expressed in human placentas, but its dynamic change and function at the human feto-maternal interface during placenta-tion remain to be elucidated. In this present study, the spatial and temporal expression and cellular localization of ADAM19 in normal human placentas were first demonstrated, and the effects of ADAM19 on trophoblast cell adhesion and invasion were further investigated by using a human choriocarcinoma cell line (JEG-3) as an in vitro model. The data demonstrated that ADAM19 was widely distributed in villous cytotrophoblast cells, syncytiotrophoblast cells, column trophoblasts, and villous capillary endothelial cells during early pregnancy. The mRNA and protein level of ADAM19 in placentas was high at gestational weeks 8—9, but diminished significantly at mid- and term pregnancy. In JEG-3 cells, the overexpression of ADAM19 led to diminished cell invasion, as well as increases in cell adhesiveness and the expression of E-cadherin, with no changes in b-catenin expression observed. These data in-dicate that ADAM19 may participate in the coordinated regulation of human trophoblast cell behaviors during the process of placentation.     

Expression of P450 aromatase and 17beta-hydroxysteroid dehydrogenase type 1 at fetal-maternal interface during tubal pregnancy

Steroidogenesis in the placenta has been studied widely, but little is known about steroid metabolism in ectopic pregnancy. Previous studies have indicated that trophoblast invasion and placentation in the uterus and the fallopian tube may be controlled by similar mechanisms. As far as 17β-estradiol (E₂) production is concerned, it has been well demonstrated that its biosynthesis in the placenta involves the action of P450 aromatase (P450arom) and 17β-hydroxysteroid dehydrogenase type 1 (17HSD1). The purpose of this study was to characterize the expression pattern of P450arom and 17HSD1 at the fetal–maternal interface, particularly in various trophoblast cells, in tubal pregnancy. Using in situ hybridization, P450arom mRNA was localized in syncytiotrophoblast (ST) cells, which are mainly responsible for hormone production during pregnancy, whereas no signal was detected in villous cytotrophoblast (VCT), column CT and extravillous CT (EVCT) cells. Immunohistochemical assays revealed that 17HSD1 is present in ST cells, a large portion of EVCT cells and 20% of column CT cells. On the other hand, no expression of 17HSD1 was detected in VCT cells. In addition, 17HSD1 was found in epithelial cells of the fallopian tube. Interestingly, the expression level of 17HSD1 in fallopian tube epithelium during tubal pregnancy was significantly higher than that during normal cycle. Our data provide the first evidence that normal and tubal pregnancies possess identical expression of P450arom and 17HSD1 in ST cells and therefore, similar E₂ production in the placenta. Further, the association of 17HSD1 with EVCT cells indicates that 17HSD1 perhaps play a role in trophoblast invasion. Finally, increased expression of 17HSD1 in epithelial cells of fallopian tube may lead to a local E₂ supply sufficient for the maintenance of tubal pregnancy.     

The chemokines, CX3CL1, CCL14, and CCL4, promote human trophoblast migration at the feto-maternal interface

Human embryo implantation is a complex process involving blastocyst attachment to the endometrial epithelium and subsequent trophoblast invasion of the decidua. Chemokines, critical regulators of leukocyte migration, are abundant in endometrial epithelial and decidual cells at this time. We hypothesized that endometrial chemokines stimulate trophoblast invasion. Chemokine receptors CX3CR1 and CCR1 were immunolocalized in human first-trimester implantation sites, specifically to endovascular extravillous trophoblasts, but not to the invading interstitial EVTs (iEVTs), with weak staining also on syncytium. CCR3 was localized to invading iEVTs and to microvilli on the syncytial surface. Expression of CX3CL1 (fractalkine), CCL7 (MCP-3), and their receptors (CX3CR1, CCR1, CCR2, CCR3, and CCR5) mRNA was examined in cellular components of the maternal-embryonic interface by RT-PCR. Both chemokines were abundant in entire endometrium and placenta, endometrial cells (primary cultures and HES, a human endometrial epithelial cell line) and trophoblast cell lines (JEG-3, ACIM-88, and ACIM-32). Chemokine receptor mRNA was expressed by placenta and trophoblast cell lines: CCR1 by all trophoblast cell types, whereas CCR2, CCR3, and CX3CR1 were more variable. CX3CR1, CCR1, CCR2, and CCR5 were also expressed by endometrial cells. Migration assays used the trophoblast cell line most closely resembling extravillous cytotrophoblast (AC1M-88). Trophoblast migration occurred in response to CX3CL1, CCL14, and CCL4, but not CCL7. Endometrial cell-conditioned media also stimulated trophoblast migration; this was attenuated by neutralizing antibodies to CX3CL1 and CCL4. Thus, chemokines are expressed by maternal and embryonic cells during implantation, whereas corresponding receptors are on trophoblast cells. Promotion of trophoblast migration by chemokines and endometrial cell conditioned medium indicates an important involvement of chemokines in maternal-fetal communication.     

MMP-26 在人正常胎盘滋养层细胞中的表达及激活素 A 对其表达的调节

胚胎植入和胎盘形成涉及细胞外基质的降解和重建,以及细胞的增殖、凋亡、迁移和分化,基质金属蛋白酶 (MMPs) 是参与这些事件的主要蛋白水解酶系统 . MMP-26 是近年来发现的 MMPs 家族的新成员,但其功能所知甚少 . 通过半定量 RT-PCR 、免疫组织化学、荧光免疫细胞化学等手段,发现人胎盘中 MMP-26 主要定位于绒毛滋养层细胞,在绒毛间质细胞中也有少量表达 . 妊娠早期,胎盘中 MMP-26 表达水平较高,至妊娠中期降至最低,但在足月胎盘中其表达又有显著提高,提示 MMP-26 可能参与妊娠早期滋养层细胞的侵润和分娩时的胎盘剥离 . 体外培养的妊娠早期人细胞滋养层细胞能产生一定水平的 MMP-26 ,而其表达受到激活素 A 的剂量依赖性刺激,表明滋养层细胞中存在 MMP-26 表达的自分泌 / 旁分泌调节 .     

Immunohistochemical localization of leukemia inhibitory factor, interleukins 1 and 6 at the primary implantation site in the rhesus monkey

Blastocyst implantation and placentation involve localized inflammatory type of responses at and around the site of nidation. In the present study, the likely involvement of inflammatory cytokines, namely, leukemia inhibitory factor (LIF), interleukins 1 alpha and 1 beta (IL-1alpha and IL-1beta) and IL-6 at the primary implantation site of the rhesus monkey was examined immunocytochemically during lacunar (n=6) and villous (n=8) stages of gestation. Trophoblast cells and extraembryonic mesenchymal cells were immunopositive for LIF and IL-1alpha. The distribution of IL-1beta and IL-6 in trophoblast cells was low in lacunar stage samples, however, a higher degree of immunopositivity for IL-6 was observed in villous stage samples. Decidual cells were immunopositive for all the cytokines studied. In lacunar stage samples, plaque cells adjacent to implanted nidus were immunopositive for all the cytokines examined, and the degree of their immunoprecipitation increased, except that of IL-1beta, during the villous stage. Luminal and glandular epithelial cells were immunopositive for LIF, IL-1alpha, IL-1beta and IL-6 in lacunar and in villous stage samples. LIF immunopositivity was detected in endothelial cells of blood vessels within and below chorionic plate and cytotrophoblast shell, while vascular smooth muscle cells were positive for all the cytokines studied. The temporo-spatial characteristics of LIF, IL-1alpha, IL-1beta and IL-6 protein expressions in primary implantation sites of the rhesus monkey suggest that these pro-inflammatory cytokines play specific roles in regulating trophoblast cell proliferation, differentiation, invasion and associated maternal tissue remodelling during early gestation.     

sLeX/L-selectin mediates adhesion in vitro implantation model

The complex implantation process is initiated by the recognition and adhesion between the embryo and uterine endometrial epithelium. The expression and interactions between the adhesive molecules from both fetal and maternal sides are crucial for the successful implantation. In this study, we aimed to investigate the expression and adhesive function of sLeX on the trophoblasts and L-selectin on uterine epithelial cells mediated the adhesion at the fetal-maternal interface, and to further explore whether this adhesion system could induce endometrial apoptosis, using in vitro implantation model consisting of the human trophoblast cell line (JAR) and human uterine epithelial cell line (RL95-2). The results showed that sLeX was expressed on JAR cells by indirect immunofluorescence staining. After transfection of JAR cells with fucosyltransferase VII (FUT7) which is the key enzyme for sLeX synthesis, the expression of FUT7 and sLeX synthesis were increased, and the percent adhesion of trophoblast cells to RL95-2 cell monolayer was significantly increased (P?相似文献   

Temporal and spatial expression of integrins and their extracellular matrix ligands at the maternal-fetal interface in the rhesus monkey during pregnancy

The integrin and extracellular matrix protein (ECM)-mediated adhesion and invasion of the receptive maternal uterine endometrium by trophoblasts is a critical event in the complex physiological process of pregnancy. Although the process has been largely characterized in mice, the relevant mechanism in primates remains unclear. We investigated the expression patterns and dynamic alterations of integrin subunits (alpha1, alpha5, alpha6, beta1, and beta4) and their ECM ligands, such as laminin (LN), type IV collagen (Col IV), and fibronectin (FN), at the maternal-fetal interface during Gestational Days 15, 25, 50, and 100 and at full term in 20 pregnant rhesus monkeys. Immunohistochemistry and in situ hybridization revealed that a relatively high expression of integrins occurred in trophoblast cells at Gestational Day 15, with the peak level occurring at Day 25. The expression level decreased from Day 50 to term. Along the invasive pathway, expression levels of integrin alpha1, alpha5, and beta1 subunits were gradually elevated from the proximal to distal column, reaching peak level in the trophoblast shell, but were reduced in those invasive extravillous cytotrophoblast (EVCT) cells in contact with the decidua. Integrin alpha1, alpha5, beta1, and beta4 subunits were also highly expressed in decidual stromal cells and moderately expressed in the maternal epithelium and endothelium. Immunoreactive FN, LN, and Col IV were distributed in EVCT and decidual stromal cells and part of the uterine epithelial and endothelial cells. These data suggest that the correlated expression of integrins and their ECM ligands at the maternal-fetal interface might be involved in regulation of cell proliferation and differentiation and the counterbalanced invasion-accelerating and invasion-restraining processes in trophoblast cells during the early stage of pregnancy.     

Changing distribution of cadherins during gestation in the uterine epithelium of lizards

The uterine epithelium provides the interface between an embryo and its mother during pregnancy. Calcium-dependent cadherins are adherens junction proteins that undergo major shifts in the uterine epithelium to facilitate the communication between maternal cells and the embryonic milieu during implantation in mammals. They are, therefore, important in trophoblast invasion and the maintenance of pregnancy. We investigated spatiotemporal changes of cadherins throughout pregnancy in the uterine epithelium of two viviparous skinks and one oviparous population, which all exhibit a noninvasive (epitheliochorial) placenta. Cadherins were identified for the first time in squamate reptiles. In all species, cadherins are reduced in the uterine epithelium as gestation progresses, which would lessen the attachment between uterine epithelial cells and allow them to stretch to accommodate embryonic growth. Interestingly, cadherins were reduced sooner after ovulation in the oviparous species than in the viviparous species. In viviparous species, the different expression of cadherins between barren and pregnant uteri from the same mother indicates that expression of cadherins may not be driven solely by maternal hormones, but also by the presence of an embryo. The redistribution of cadherins in squamates is comparable to that of mammals, reflecting establishment of feto-maternal communication during the peri-implantation period. As there is no breaching of maternal tissue in lizards, the change in adherens junctional properties are thus not exclusive to mammals with invasive placentae, which suggests that similar molecular mechanisms regulate changes to uterine epithelia during pregnancy across placental types.     

Early stages of trophoblastic invasion of the maternal vascular system during implantation in the macaque and baboon.

Trophoblastic invasion and remodeling of the uteroplacental (spiral) arteries in primates are well-documented, but virally nothing is known of the early stages of these phenomena. Therefore, we examined invasion of the maternal vasculature in macaques and baboons at, and immediately following, implantation. Following penetration of the uterine epithelium (day 9), trophoblast spreads along the residual epithelial basal lamina. By day 10, cytoplasmic processes penetrate the epithelial and endothelial basal laminae, and syncytial trophoblast insinuates itself between maternal endothelial cells. As lacunae develop, both syncytial and cytotrophoblast are exposed to maternal blood. Endovascular cytotrophoblast was first observed in subepithelial dilated capillaries and venules. These vessels are lined by increasingly hypertrophied endothelial cells. The spiral arterioles are unmodified at this time. Particularly interesting was the observation that there is rapid extensive endovascular trophoblast invasion of the spiral arterioles immediately beneath the implantation site. By day 14-16 nearly all of the small arterioles directly beneath the site are completely occluded. There is no invasion of the veins in this region. Somewhat later, the deeper arterioles in the principal zone are invaded. Rather than a continuous stream of cells invading the deeper arterioles, these endovascular cells occur in clusters ranging from a few cells to groups of cells that completely plug the lumen. Our results indicate that trophoblastic invasion of maternal vessels occurs very early; and, at least initially, trophoblast can migrate between and along endothelial cells without causing their lysis. The endovascular cells eventually interrupt the endothelial lining of the arterioles and penetrate the walls of the vessels. The occlusion of arterioles underneath the site suggests that circulation through the lacunae at this stage is indirect. Corresponding stages of human development were examined, and no invasion of arterioles could be observed prior to formation of an extensive cytotrophoblastic shell.     

Formation of atypical podosomes in extravillous trophoblasts regulates extracellular matrix degradation

Throughout pregnancy the cytotrophoblast, the stem cell of the placenta, gives rise to the differentiated forms of trophoblasts. The two main cell lineages are the syncytiotrophoblast and the invading extravillous trophoblast. A successful pregnancy requires extravillous trophoblasts to migrate and invade through the decidua and then remodel the maternal spiral arteries. Many invasive cells use specialised cellular structures called invadopodia or podosomes in order to degrade extracellular matrix. Despite being highly invasive cells, the presence of invadapodia or podosomes has not previously been investigated in trophoblasts. In this study these structures have been identified and characterised in extravillous trophoblasts. The role of specialised invasive structures in trophoblasts in the degradation of the extracellular matrix was compared with well characterised podosomes and invadopodia in other invasive cells and the trophoblast specific structures were characterised by using a sensitive matrix degradation assay which enabled visualisation of the structures and their dynamics. We show trophoblasts form actin rich protrusive structures which have the ability to degrade the extracellular matrix during invasion. The degradation ability and dynamics of the structures closely resemble podosomes, but have unique characteristics that have not previously been described in other cell types. The composition of these structures does not conform to the classic podosome structure, with no distinct ring of plaque proteins such as paxillin or vinculin. In addition, trophoblast podosomes protrude more deeply into the extracellular matrix than established podosomes, resembling invadopodia in this regard. We also show several significant pathways such as Src kinase, MAPK kinase and PKC along with MMP-2 and 9 as key regulators of extracellular matrix degradation activity in trophoblasts, while podosome activity was regulated by the rigidity of the extracellular matrix.     

Murine abortion is associated with enhanced interleukin-6 levels at the feto-maternal interface

CBA/JXDBA/2J murine abortion is known to be associated with increased local and peripheral Th1-cytokines levels. The role of the pro-inflammatory interleukin-6 (IL-6) in murine abortion remains unclear. In humans, IL-6 was reported to be elevated at the onset of spontaneous abortion. The aim of our study was to evaluate the levels of IL-6 during murine pregnancy in (1) the normal murine pregnancy combination CBA/JXBALB/c and in (2) the CBA/JXDBA/2J abortion prone mating combination. We measured IL-6 serum levels by ELISA and local (placental and decidual) IL-6 levels by flow cytometry and immunohistochemistry. The expression of the IL-6 receptor gp80 was further analyzed. We additionally evaluated the number of mast cells and macrophages at the feto-maternal interface as a putative IL-6 source in reproductive tissues. IL-6 and gp80 were expressed in decidual cells as well as in different trophoblast types. Flow cytometry analysis showed increased numbers of IL-6+ cells in abortion placentas and deciduas compared to control pregnant mice. We observed an elevated number of mast cells and macrophages at the feto-maternal interface from abortion mice in comparison to control mice. Interestingly, we found very high numbers of mast cells, macrophages and IL-6+ cells in resorption tissue compared to control tissues. Flow cytometry studies confirmed that macrophages are being an important source of IL-6 at the feto-maternal interface. The mRNA IL-6 levels were also enhanced in placenta and decidua from mice with high abortion rate compared to normal pregnant mice, as analyzed by RT-PCR. Our results suggest that IL-6 produced not only by immunocompetent cells such as macrophages and mast cells, but also by trophoblasts and decidua cells, is directly involved in the pathology of abortion.     

Calcitonin gene-related peptide (CALCA) is a proangiogenic growth factor in the human placental development

Recent studies have shown that homozygous knockout of gene for calcitonin gene-related peptide (CALCA) receptor component, calcitonin receptor-like receptor (CALCRL), led to extreme hydrops fetalis and embryonic death, underlining the critical role of CALCA in embryonic development and fetal growth. The present study was designed to determine the cellular localization of CALCA and its receptor components, CALCRL and receptor activity modifying protein 1 (RAMP1), at the human implantation site during early pregnancy; to assess whether CALCA regulates in vitro angiogenesis of human endothelial cells; and to examine whether CALCA can improve angiogenic imbalance in preeclamptic placental explants. Our studies demonstrated that both protein and mRNA for CALCA were expressed by the villous and extravillous trophoblasts and decidual cells in the first-trimester villous tissues. CALCA receptor components, CALCRL and RAMP1, were expressed by both villous and extravillous trophoblast cells, as well as vascular endothelial cells. CALCA induced both endothelial proliferation and migration in a dose- and time-dependent manner, and it promoted capillarylike tube formation of human umbilical vein endothelial cells (HUVECs) on Matrigel. CALCA-induced angiogenesis of human endothelial cells was completely blocked by CALCA antagonist CALCA(8-37). Further, conditioned medium from preeclamptic placental explants significantly inhibited HUVEC capillarylike tube formation compared with gestational age-matched controls, and conditioned medium from preeclamptic placental explants incubated with CALCA significantly improved capillarylike tube formation. We conclude that CALCA induces in vitro angiogenesis by stimulating endothelial cell proliferation, migration, and capillarylike tube formation; thus, CALCA at the human implantation site may constitute a potential autocrine or paracrine mechanism that could modify placental angiogenesis and neovascularization.     

Blastocyst implantation in the Chinese hamster (Cricetulus griseus)

Embryonic development of the Chinese hamster (Cricetulus griseus) was studied from the onset of implantation to the formation of the parietal yolk sac placenta. Implantation began on day 6 of pregnancy, when the embryo became fixed to the uterine luminal epithelium. At this time there was no zona pellucida, and microvilli of the trophoblast and uterine epithelium were closely apposed. Stromal cells immediately adjacent to the implantation chamber began to enlarge and accumulate glycogen. By day 7 the mural trophoblast penetrated the luminal epithelium in discrete area. The trophoblast appeared to phagocytize uterine epithelial cells, although epithelium adjoining the points of penetration was normal. In other areas nascent apical protrusions from the uterine epithelium indented the surface of the trophoblast. The epiblast had enlarged and both visceral and parietal endoderm cells were present. The well-developed decidual cells were epithelioid and completely surrounded the implantation chamber. On day 8 the uterine epithelium had disappeared along the mural surface of the embryo. The embryonic cell mass was elongated and filled the yolk sac cavity. Reichert's membrane was well developed. The uterine epithelial basal lamina was largely disrupted, and the trophoblast was in direct contact with decidual cells. Primary and secondary giant trophoblast cells were present and in contact with extravasated maternal blood. The mural trophoblast formed channels in which blood cells were found in close proximity to Reichert's membrane. Decidual cells were in contact with capillary epithelium and in some cases formed part of the vessel wall. Structural changes occurring in the embryo and endometrium during implantation in the Chinese hamster are described for the first time in this report and are compared to those described for some other myomorph rodents.