Trophoblast Adhesion Research Articles

The role of leukemia inhibitory factor in tubal ectopic pregnancy

IntroductionEctopic pregnancy is unique to humans and a leading cause of maternal morbidity and mortality. The etiology remains unknown however factors regulating embryo implantation likely contribute. Leukemia inhibitory factor (LIF) has roles in extravillous trophoblast adhesion and invasion and is present in ectopic implantation sites. We hypothesised that LIF facilitates blastocyst adhesion/invasion in the Fallopian tube, contributing to ectopic pregnancy. MethodsWe immunolocalised LIF receptor (R) in tubal ectopic pregnancy (N = 5). We used an oviduct cell line (OE-E6/E7) to model Fallopian tube epithelial cells and a trophoblast spheroid co-culture model (HTR-8/SVneo cell line formed spheroids) to model blastocyst attachment to the Fallopian tube. We examined LIF signaling pathways in OE-E6/E7 cells by Western blot. The effect of LIF and LIF inhibition (using a novel LIF inhibitor, PEGLA) on first-trimester placental outgrowth was determined. ResultsLIFR localised to villous and extravillous trophoblast and Fallopian tube epithelium in ectopic pregnancy. LIF activated STAT3 but not the ERK pathway in OE-E6/E7 cells. LIF stimulated HTR-8/SVneo spheroid adhesion to OE-E6/E7 cells which was significantly reduced after PEGLA treatment. LIF promoted placental explants outgrowth, while co-treatment with PEGLA blocked outgrowth. DiscussionOur data suggests LIF facilitates the development of ectopic pregnancy by stimulating blastocyst adhesion and trophoblast outgrowth from placental explants. Ectopic pregnancy is usually diagnosed after 6 weeks of pregnancy, therefore PEGLA may be useful in targeting trophoblast growth/invasion. ConclusionLIF may contribute to the development of ectopic pregnancies and that pharmacologically targeting LIF-mediated trophoblast outgrowth may be useful as a treatment for ectopic pregnancy.

Human placental multipotent mesenchymal stromal cells modulate trophoblast migration via Rap1 activation

IntroductionLittle is known about the interaction between human placental multipotent mesenchymal stromal cell (hPMSC) and trophoblast. We hypothesize that hPMSCs produce hepatocyte growth factor (HGF) which may interact with trophoblasts and regulate their migration during placentation. MethodshPMSCs were isolated from term placentas and conditioned medium was collected after 2 days of culture in hypoxic (<1% O2) or control (20% O2) conditions. Selective agonist and inhibitor or siRNA for protein kinase A (PKA) or Rap1 were combined with Rap1-GTP pull down assays, flow cytometry, integrin β1 activation assays and adhesion and migration studies to investigate HGF signaling effects in trophoblasts. The hPMSC abundance and HGF level in preeclamptic placentas were compared with gestational age-matched controls. ResultsHGF was expressed by hPMSCs and was decreased in hypoxia. HGF induced cAMP production and Rap1 activation in trophoblasts, which in turn activated integrin β1. The HGF and PKA activator 6-Bnz-cAMP induced Rap1 activation with increased trophoblast adhesion and migration. The alterations were inhibited by PKA inhibitor H89 or Rap1 siRNA. HGF and cAMP expression were reduced in preeclamptic placentas. hPMSC number was decreased in preeclamptic placenta compared to controls (0.68 ± 0.1% vs. 1.32 ± 0.5%; P = 0.026). hPMSC conditioned medium enhanced trophoblast migration which was inhibited by c-Met blocking antibody, but migration was reduced by conditioned medium from hPMSC cultured in hypoxia. ConclusionshPMSCs secrete HGF and increase trophoblast cAMP production. The cAMP effector PKA modulates adhesion and migration of trophoblast via signaling to Rap1 and integrin β1.

Autocrine and Paracrine Mechanisms of Prostaglandin E2 Action on Trophoblast/Conceptus Cells through the Prostaglandin E2 Receptor (PTGER2) during Implantation

The conceptus and endometrium secrete large amounts of prostaglandin E₂ (PGE₂) into the porcine uterine lumen during the periimplantation period. We hypothesized that PGE₂ acts on conceptus/trophoblast cells through auto- and paracrine mechanisms. Real-time RT-PCR analysis revealed that PGE₂ receptor (PTGER)2 mRNA was 14-fold greater in conceptuses/trophoblasts on days 14-25 (implantation and early placentation period) vs preimplantation day 10-13 conceptuses (P < .05). Similarly, expression of PTGER2 protein increased during implantation. Conceptus expression of PTGER4 mRNA and protein did not differ on days 10-19. PGE₂ stimulated PTGER2 mRNA expression in day 15 trophoblast cells through PTGER2 receptor signaling. PGE₂ elevated aromatase expression and estradiol-17β secretion by trophoblast cells. Moreover, PGE₂ and the PTGER2 agonist, butaprost, increased the adhesive capacity of both human HTR-8/SVneo trophoblast and primary porcine trophoblast cells to extracellular matrix. This PGE₂-induced alteration in trophoblast cell adhesion to extracellular matrix was abolished by incubation of these cells with AH6809 (PTGER2 antagonist), ITGAVB3-directed tetrapeptide arg-gly-asp-ser or integrin ITGAVB3 antibody. PGE₂ stimulated adhesion of porcine trophoblast cells via the estrogen receptor and MEK/MAPK signaling pathway. PGE₂ induced phosphorylation of MAPK1/MAPK3 through PTGER2 and up-regulated expression of cell adhesion proteins such as focal adhesion kinase and intercellular adhesion molecule-1. Our study indicates that elevated PGE₂ in the periimplantation uterine lumen stimulates conceptus PTGER2 expression, which in turn promotes trophoblast adhesion via integrins, and synthesis and secretion of the porcine embryonic signal estradiol-17β. Moreover, the mechanism through which PGE₂ increases trophoblast adhesion is not species specific because it is PTGER2- and integrin-dependent in both porcine and human trophoblast cells.

HPV-16 exposed mouse embryos: a potential model for pregnancy wastage

Placentas from spontaneous abortions and preterm deliveries have a higher prevalence of Human papillomavirus (HPV) compared to placentas from elective abortions and term births. The objective was to determine the effects of HPV-16 on the adhesion and implantation properties of early embryo trophoblasts. Two-cell mouse embryos were cultured (medium G2, 5 % CO2, 37 °C) for 72-96 h and exposed to either HPV-16 rich SiHa cell lysates which were refrigerated after mechanical lysis, thawed lysates which had been frozen for freeze/thaw lysis method, or control medium, incubated (4-5 days) and evaluated by microscopy (N = 96 embryos, 3 repeated experiments). Trophoblasts were stained and images were digitized. Adhesion and dimension data were analyzed by Chi-square and t test, respectively. HPV-16 exposed embryos exhibited less adhesion through reduced implantation compared with the control (combined lysates 53.8 vs. 85.7 %, P < 0.05). Refrigerated and thawed lysate groups had similar reduced implantations (58.3 vs. 50.0 %). Of the embryos with implantation, 100 % in the refrigerated lysates were noted to have loose or abnormal adhesion. This was measured when embryos were noted to be lost after washes with HTF. There was no difference in trophoblast viability among the groups. Total trophoblast area was greater in the HPV-16 exposed frozen lysate group (1,881.8 ± 605.3 vs. control 848.8 ± 298.0 square units, mean ± SEM). HPV-16 inhibited trophoblasts adhesion needed for normal implantation, but not embryo development. Total trophoblast spread was increased after HPV-16 exposure suggesting that HPV-16 altered trophoblast migration. These results suggest that HPV-16 may induce abnormal placental growth resulting in pregnancy wastage.

Subcellular localization of L-selectin ligand in the endometrium implies a novel function for pinopodes in endometrial receptivity

BackgroundApical surfaces of human endometrial epithelium and endothelium are key elements for the initiation of molecular interactions to capture the blastocyst or leukocyte, respectively. The L-selectin adhesion system has been strongly proposed to play an important role in the initial steps of trophoblast adhesion and promotion of integrin-dependent processes, ultimately culminating in the establishment of the embryo-maternal interface. On the basis of these facts, we hypothesized a novel role for pinopodes as the first embryo-fetal contact sites to contain the highest subcellular expression of L-selectin ligand suggesting its role in early adhesion as predicted. Thus, the objective of this study was therefore to determine the subcellular pattern of distribution of the L-selectin ligand (MECA-79) in human endometrial apical membrane region during the window of implantation.MethodsEndometrial biopsies of secretory phases from fertile females ranging in age between 25 and 42years were studied using several approaches, including scanning electron microscopy (SEM), immunostaining for light microscopy and transmission electron microscopy (TEM), and immunoblotting as well as statistical analysis of the area-related numerical densities of immunoreactive MECA-79-bound nanogolds to detect the expression pattern and the subcellular distribution pattern of L-selectin ligand (MECA-79) in human endometrium during the window of implantation.ResultsThe endometrial biopsies were scored according the dating criteria of Noyes et al. by an experienced histologist. The SEM images of the midluteal phase specimens revealed that fully developed pinopodes were abundant in our samples. HRP-immunostaining and immunofluorescent staining as well as immunoblotting revealed that MECA-79 was expressed in the midluteal phase specimens. The results of immunogold TEM illustrated the expression of MECA-79 in human pinopodes in the midluteal phase and a higher area-relate numerical density in pinopodes compared to that of the uterodome-free areas.ConclusionsThis is the first demonstration of the subcellular localization of MECA-79 in the human pinopodes which may indicate a novel role for pinopodes to be capable of shear-stress-dependent tethering-type adhesion in the initial phases of human embryo implantation.

An Alternative Role of C1q in Cell Migration and Tissue Remodeling: Contribution to Trophoblast Invasion and Placental Development

Fetal trophoblast cells invading the decidua in the early phase of pregnancy establish complex interaction with the maternal extracellular matrix. We discovered that C1q was widely distributed in human decidual stroma in the absence of C4 and C3 and was actively synthesized by migrating extravillous trophoblasts. The cells expressed the messages for the three chains of C1q and secreted this complement component that interacted with the proteins of the decidual extracellular matrix. Solid phase-bound C1q promoted trophoblast adhesion and migration, and cell binding to C1q resulted in activation of ERK1/2 MAPKs. Ab inhibition experiments showed that the receptors for the globular head of C1q/p33 and α(4)β(1) integrin were both involved in this process and were colocalized on the cell surface following binding of C1q to trophoblasts. We also found that C1q(-/-) mice manifested increased frequency of fetal resorption, reduced fetal weight, and smaller litter sizes compared with wild-type mice. C1q deficiency was associated with impaired labyrinth development and decidual vessel remodeling. Collectively, these data suggest that C1q plays an important role in promoting trophoblast invasion of decidua and that defective local production of C1q may be involved in pregnancy disorders, such as pre-eclampsia, characterized by poor trophoblast invasion.

Extracellular matrix remodelling in rat endometrium during early pregnancy: The role of fibronectin and laminin

The endometrial extracellular matrix (ECM) remodelling has a crucial role in the establishment of a successful pregnancy. In addition to its basic function such as regulation of cell function, differentiation, migration, proliferation, the substantial alterations in the endometrial ECM may play a specific role in the trophoblast invasion, placentation, cell death and formation of the proper and functional implantation chamber around the embryo. In the present study, immunolocalizations of fibronectin and laminin were determined using avidin–biotin complex–peroxidase in rat implantation sites during 7–10 days of pregnancy. Both proteins were present in the basal membrane of blood vessels and in decidual matrix whereas they were absent or had very weak reactivity in the primary decidual zone on day 7. When placentation has begun, the immunoreactivity of both proteins was increased in the placental bed and in the basal membrane of blood vessels of the mesometrial region. The immunolocalization of both proteins seemed to be decreased in the antimesometrial decidua, however, it was increased in the mesometrial decidual matrix on days 9 and 10. Therefore, it could be suggested laminin and fibronectin demonstrating dynamic expressions in relation with the morphological differentiation of endometrial stroma may play crucial roles in the control of trophoblast adhesion and invasion, in placentation and angiogenesis, in the determination of cell shape and fate thus contributing the endometrial receptivity and a successful pregnancy.

Adhesion behaviors of human trophoblast cells by contact with endothelial cells

Although it is still not clear whether migratory trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by a mechanism of directional cell division/proliferation, this process involves the attachment and adhesion of trophoblasts to endothelial cells lining the blood vessel walls. This raises the possibility that the cell–cell contact with endothelial cells may regulate trophoblast cell adhesion behaviors according to the surrounding flow condition. To test this, the adhesion forces of early gestation human trophoblast cells (TCs) cultured on glass slides coated with type I rat collagen or cultured with human umbilical vein endothelial cells (HUVECs) were measured quantitatively using a micropipette aspiration technique. Then, the resistance of TCs co-cultured with HUVECs to flow-induced shear stress was assessed with a flow chamber technique. The results showed that the adhesion force of TCs to glass slides coated with collagen was positively correlated with the concentration of collagen. By contact with endothelial cells, the adhesion force and the resistance to shear stress for the TCs were significantly enhanced. The interdiction of integrin β1 interaction remarkably reduced the adhesion forces of TCs to endothelial cells, hence their resistance to shear stress. The results therefore suggest that the contacts of TCs with endothelial cells enhance the adhesion forces of human TCs, partially by regulating with the integrin β1 according to the flow condition (i.e., the shear stress) in such a way to prevent the TCs from being carried downstream by flowing blood.

Interleukin-11 and Leukemia Inhibitory Factor Regulate the Adhesion of Endometrial Epithelial Cells: Implications in Fertility Regulation

Embryo implantation requires the closely harmonized processes of apposition, attachment, and adhesion of the conceptus to the maternal endometrial epithelium. IL-11 and leukemia inhibitory factor (LIF), two IL-6 family cytokines, are produced by the endometrium and are absolutely required for implantation in mice. We examined the effect of IL-11 and LIF on human endometrial epithelial cell adhesion. Both cytokines increased adhesion of primary human endometrial epithelial cells to fibronectin and collagen IV. IL-11 stimulated, whereas LIF had no effect on the adhesion of trophoblast to endometrial epithelial cells. Focused oligogene arrays were used to identify extracellular matrix and adhesion molecules mRNAs regulated by endometrial epithelial cells. We demonstrated by real-time RT-PCR and antibody arrays that both cytokines increased integrin-alpha2 mRNA and protein by endometrial epithelial cells. Signal transducers and activators of transcription (STAT)-3 inhibition reduced IL-11- and LIF-mediated epithelial cell adhesion to fibronectin, suggesting both cytokines regulated adhesion via phosphorylation of STAT3. Addition of either IL-11 neutralizing antibody and IL-11 or LIF and LIF antagonist to endometrial epithelial cells abolished cytokine induced phosphorylated STAT3. LIF but not IL-11 induced adhesion to collagen IV was reduced by an integrin-alpha2beta1 neutralizing antibody. This study demonstrated that IL-11 and LIF regulated endometrial epithelial cell adhesion, suggesting that targeting IL-11 and LIF may be useful in regulating fertility by either enhancing or blocking implantation.

525. ROLES FOR HUMAN CHORIONIC GONADOTROPHIN IN EMBRYO-ENDOMETRIAL CROSS-TALK DURING BLASTOCYST IMPLANTATION

Emerging evidence suggests an important role for the early embryo product human chorionic gonadotrophin (hCG) in embryo-endometrial interactions critical for successful embryo implantation1. The human endometrium is also a source of hCG, with maximal expression of hCG and its receptor, hCG/LHR, in endometrial epithelial cells during the window of implantation in vivo2,3, and in primary endometrial epithelial cells (EECs)3. Implantation is tightly regulated by growth and regulatory factors produced within the embryo-endometrial microenvironment. We hypothesise that embryo/endometrial-derived hCG mediates the molecular cross talk vital for successful implantation. The main objective of this study was to investigate the effect of hCG on the production of a selected cohort of 42 cytokines and growth factors by EECs. These included those with both known and previously unidentified roles during implantation. The secretory profile of cytokines/growth factors produced by EECs was also analysed. EECs (n=8 cultures) were isolated from biopsies collected from fertile cycling women. Cells were treated without or with recombinant hCG for 48 hr and conditioned media collected for quantitative analysis using LuminexTM multiplex technology. For the first time, a secretory profile of 42 cytokines and growth factors produced by EECs was established, as was the identification of fibroblast growth factor-2 (FGF-2) secretion by human endometrial epithelium. hCG (2 IU/ml) significantly increased the production of a number factors including those with known roles during trophoblast migration and adhesion (CX3CL1; 71±31%, CXCL10; 67±24%, CCL4; 87±12%), in trophoblast differentiation (IL-1α ; 68±31%) and with unidentified roles during implantation (CCL22; 78±40%, GM-CSF; 45±16%, FGF-2; 50±25%; all p&lt;0.05). Upregulation of the known hCG regulated proteins, VEGF and LIF, validated this study. These findings clearly support roles for the embryo/endometrium via hCG in actively contributing to the molecular cross-talk during the early stages of implantation.

The Macaque Placenta—A Mini-Review

As part of an overview of the female reproductive organs in the macaque monkey, the present paper presents normal placental development. Although normally not examined in routine toxicologic pathology, the interest in the macaque as a model for reprotoxicity studies is increasing significantly. Based on different classifications, the macaque placenta belongs to the chorioallantoic, (bi)discoid, villous, deciduate, and hemochorial placental type. Within the first fourteen days after fertilization, a large number of events subsequently occur (apposition, adhesion, penetration and traversal of trophoblasts, blood vessel penetration, and development of villi). After this period, the basic placental structure has been laid down in the endometrium, and the initial communication between mother and fetus has been established. Further expansive growth of the placenta and development of anchoring villi are believed to be accomplished by continuous proliferation and migration of the trophoblasts from the trophoblastic shell. Despite the same function of human and macaque placentas, the morphologic structure and developmental timelines are different. Possible toxicological and physiological implications of these differences toward the value of macaques within reprotoxicity studies is discussed at the end of this paper. Besides a transporting role between mother and fetus, the placenta is also an endocrine organ that synthesizes a variety of hormones and cytokines. They influence ovarian and uterine physiology at the start of pregnancy and fetal and mammary physiology during gestation and around labor, respectively. Competing Interests: This article was sponsored by Covance Inc. and Schering-Plough. Eveline P. C. T. de Rijk and Eric Van Esch are employed by Schering-Plough. No other competing interests were declared.

Leukemia inhibitory factor promotes human first trimester extravillous trophoblast adhesion to extracellular matrix and secretion of tissue inhibitor of metalloproteinases-1 and -2

BACKGROUNDLeukemia inhibitory factor (LIF) is a pleiotropic cytokine that is essential for blastocyst implantation in mice. It has been suggested that LIF may play a role in human first trimester extravillous trophoblast (EVT) invasion. The aim of the present study was to establish whether LIF induces changes in EVT function related to invasiveness.METHODSPrimary first trimester human EVT cell cultures were treated with/without LIF and the effects on cell adhesion to fibronectin (FN), vitronectin (VN) and laminin (LN) were assessed. Transcript levels of integrin subunits that mediate cell adhesion to these extracellular matrix (ECM) elements were determined by real-time RT–PCR. Matrix metalloproteinase (MMP)2 and MMP9 secretion was assessed by gelatine zymography and tissue inhibitors matrix metalloproteinase (TIMP) -1 and TIMP-2 secretion by enzyme-linked immunosorbent assay.RESULTSEVT cells showed increased adhesion to FN, VN and LN ECM elements in response to LIF (20, 20 and 29%, respectively, P < 0.05 FN and VN compared to control; and P < 0.001 LN compared to control). Integrin β4 mRNA levels decreased by 50% following LIF treatment (P < 0.001 versus control). MMP2 and MMP9 secretion was not affected by LIF but LIF did increase secretion of TIMP-1 and -2 (P < 0.001 versus control). LIF stimulated the phosphorylation of signal transducer and activator of transcription (STAT) 3 protein while it did not affect STAT3 protein abundance. The addition of a LIF inhibitor attenuated the LIF-induced STAT3 phosphorylation in EVT.CONCLUSIONThe results suggest that LIF can regulate EVT invasion, suggesting an important role in early placental development.

CX3CL1 and CCL14 Regulate Extracellular Matrix and Adhesion Molecules in the Trophoblast: Potential Roles in Human Embryo Implantation1

Embryo implantation is a complex process involving blastocyst attachment to the endometrial epithelium and subsequent trophoblast invasion of the decidua. We have previously shown that the chemokines CX3CL1 and CCL14 are abundant in endometrial vasculature, epithelial, and decidual cells at this time, and that their receptors, CX3CR1 and CCR1, are present on invading human trophoblasts. CX3CL1 and CCL14 promote trophoblast migration. We hypothesized that these endometrial chemokines promote trophoblast migration by regulating adhesion molecules and extracellular matrix (ECM) components on the trophoblast, similar to mechanisms used in leukocyte trafficking. Trophoblast cells (AC1M-88) used previously showed a marked increase in adhesion to fibronectin following treatment with CX3CL1 and CCL14. Alterations in trophoblast adhesion and ECM following chemokine stimulation were examined using pathway-specific oligo-arrays and quantitative real-time RT-PCR. More than 30 genes were affected by CX3CL1 treatment, and 15 genes were found to be regulated by CCL14 treatment. Real-time RT-PCR quantitation revealed significant changes in the mRNA transcripts of alpha-catenin (CTNNA1), extracellular matrix protein 1 (ECM1), osteopontin (SPP1), integrin alpha 6 (ITGA6), matrix metalloproteinase 12 (MMP12), and integrin beta 5 (ITGB5) following chemokine treatment. Several of these genes have previously been implicated in implantation. Immunohistochemistry confirmed the presence of integrin alpha 6 and SPP1 protein in first-trimester human implantation sites. The temporal and spatial expression of chemokines, their receptors, adhesion, and ECM at the maternal-fetal interface emphasizes an important role in the controlled directional migration of trophoblasts through the maternal decidua. For the first time, this study demonstrates the direct effects of CX3CL1 and CCL14 on trophoblast adhesion molecules and ECM, suggesting mechanisms by which trophoblast cells migrate during early pregnancy.

Decidual endothelial cells express surface-bound C1q as a molecular bridge between endovascular trophoblast and decidual endothelium

This study was prompted by the observation that decidual endothelial cells (DECs), unlike endothelial cells (ECs) of blood vessels in normal skin, kidney glomeruli and brain, express surface-bound C1q in physiologic pregnancy. This finding was unexpected, because deposits of C1q are usually observed in pathologic conditions and are associated with complement activation. In the case of DECs, we failed to detect immunoglobulins and C4 co-localized with C1q on the cell surface. Surprisingly, DECs expressed mRNA for the three chains of C1q and secreted detectable level of this component in serum-free medium. The ability to synthesize C1q is acquired by DECs during pregnancy and is not shared by ECs obtained from endometrium and from other sources. Cell-associated C1q has a molecular weight similar to that of secreted C1q and is released from DECs following treatment with heparinase or incubation at low pH. This suggests that C1q binds to DECs and it is not constitutively expressed on the cell surface. C1q is localized at contact sites between endovascular trophoblast and DECs and acts as an intercellular molecular bridge because adhesion of endovascular trophoblast to DECs was inhibited by antibodies to C1q and to a receptor recognizing its globular portion expressed on trophoblast.

213. Chemokines: key players at the maternal - fetal interface

Establishment of pregnancy requires extensive communication at the maternal-fetal interface and involves a plethora of locally acting molecules, including the chemokines. Chemokines are multifunctional molecules initially described for roles in leukocyte trafficking, but since found to participate in many other processes such as differentiation and directed migration. Previously we have shown that the chemokines, CX3CL1 and CCL14, are abundant in human endometrial vasculature, leukocytes, epithelial and decidual cells at the time of implantation and that their receptors, CX3CR1 and CCR1, are present on invading human trophoblast. CX3CL1 and CCL14 directly promote human trophoblast migration. We hypothesised that these endometrial chemokines promote trophoblast migration by regulating adhesion molecules and extracellular matrix (ECM) components on the trophoblast, similar to mechanisms used in leukocyte trafficking. Trophoblast cells (AC1M-88) used previously, showed a marked increase in adhesion to fibronectin following treatment with CX3CL1 and CCL14. Alterations in trophoblast adhesion associated and ECM genes following chemokine stimulation were examined using pathway specific oligo-arrays and quantitative real-time RT–PCR. Over 30 transcripts were affected by CX3CL1 treatment and 15 were regulated by CCL14 treatment. Real-time RT–PCR confirmed significant changes in the mRNA transcripts of α-catenin (CTNNA1), extracellular matrix protein-1 (ECM1), osteopontin (SPP1), integrin α6 (ITGA6), matrix metalloproteinase-12 (MMP12) and integrin β5 (ITGB5) following chemokine treatment. Several of these molecules have previously been implicated in implantation. Immunohistochemistry confirmed the presence of integrin α6, SPP1 and ECM1 protein in first trimester human implantation sites. The temporal and spatial expression of chemokines, their receptors and adhesion related molecules at the maternal-fetal interface emphasises an important role in the controlled directional migration of trophoblast through the maternal decidua. For the first time this study demonstrates direct effects of CX3CL1 and CCL14 on trophoblast adhesion and ECM molecules suggesting mechanisms by which trophoblast cells migrate during early pregnancy.

Galectin 15 (LGALS15) functions in trophectoderm migration and attachment

Galectin 15 (LGALS15) is expressed specifically by the endometrial luminal epithelium (LE) of the ovine uterus in concert with blastocyst growth, elongation, and implantation. LGALS15 contains a predicted carbohydrate recognition domain (CRD) as well as LDV and RGD recognition sequences for integrin binding. Studies tested the hypothesis that LGALS15 is a secreted regulator of blastocyst development, as well as growth, migration, adhesion, and apoptosis of trophoblast. Bovine embryos were produced in vitro by standard conditions, and putative zygotes were cultured in the presence of recombinant ovine LGALS15. Rates of embryo cleavage and blastocyst formation were not affected by LGALS15. LGALS15 moderately increased proliferation of ovine trophectoderm (oTr) cells. Staurosporine elicited apoptosis of oTr cells, which could be partially inhibited by LGALS15. Migration of oTr cells was stimulated by LGALS15 that was dependent on Jun N-terminal kinase (JNK). A dose-dependent increase in oTr cell attachment to LGALS15 was found that could be inhibited by cyclic GRGDS, but not GRADS, peptides. Mutation of the LDVRGD integrin binding sequence of LGALS15 to LADRAD decreased its ability to promote oTr cell attachment, whereas mutation of the CRD had little effect. LGALS15 induced formation of robust focal adhesions in oTr cells that was abolished by mutation of the LDVRGD sequence. Collectively, these results support the hypothesis that LGALS15 stimulates trophectoderm cell migration and attachment via integrin binding and activation which are critical to blastocyst elongation and implantation.

SECRETED PHOSPHOPROTEIN 1 ISOLATED FROM UTERINE SECRETIONS PROMOTES INTEGRIN-MEDIATED ADHESION OF PORCINE TROPHECTODERM CELLS

In domestic pigs, over two-thirds of reproductive losses occur during the first 25 days of gestation, a period during which rapid conceptus development, trophoblast adhesion and implantation are initiated. Successful conceptus implantation requires pregnancy-specific alterations in extracellular matrix (ECM) at the conceptus-maternal interface. The pig luminal epithelium (LE) immediately adjacent to the implanting conceptus secretes SPP1 (also known as osteopontin), an ECM molecule involved in cell-cell and cell-ECM communication that is believed to mediate conceptus-uterus interactions. We hypothesized that SPP1 expressed by the uterine LE bridges to integrin receptors on conceptus trophectoderm to stimulate adhesion at the conceptus-maternal interface. The objective of this study was to determine if SPP1 isolated from uterine secretions (uterine milk) supports trophectoderm cell adhesion in vitro. To accomplish this, we employed a unilaterally pregnant sheep model, where the ovary ipsilateral to the right uterine horn was removed and a double ligature placed at the base of the right uterine horn at the bifurcation. At the following estrus, sheep were mated to generate gravid and nongravid uterine horns, hysterectomized on day 120 and the uterine milk within nongravid horns collected. SPP1 was purified from uterine milk using a DEAE-Sephacel column (ion exchange chromatography) followed by dual phenyl-Sepharose columns (hydrophobic chromatography). A 45 kDa fragment, previously identified at the apical surface of the sheep LE during pregnancy, and a 25 kDa fragment were isolated. The spontaneously immortalized pig trophectoderm cell line pTr2 was allowed to attach to serial dilutions (20 μg/ml to 40 ng/ml) of the following proteins: bovine milk SPP1 (bSPP1; purified in the manner described above), ovine uterine milk SPP1 (oSPP1), recombinant rat SPP1 containing the RGD cellbinding sequence (rSPP1-RGD), recombinant rat SPP1 containing an RAD sequence in place of the RGD sequence (rSPP1-RAD), full-length human fibronectin (FN; positive control) and BSA (negative control). Dose-dependent cell adhesion was observed for bSPP1, oSPP1 and rSPP1- RGD, all of which supported higher levels of adhesion than FN at concentrations ranging from 0.15 and 1.25 μg/ml. Only at concentrations of 2.5 μg/ml and higher did FN support greater pTr2 adhesion than the various SPP1 proteins. Cell adhesion was not supported by rSPP1-RAD over BSA controls at any concentration, strongly suggesting that adhesion to the various SPP1 proteins and FN occurred through RGD-binding integrin receptors. Integrins require the presence of divalent cations to allow ligand binding. This property was used to further confirm that pTr2 cell adhesion to SPP1 was integrin-mediated. Cells were allowed to attach in the presence of varying levels of divalent cations: 1) no cations; 2) 1 mM Ca2+ + 2 mM Mg2+; 3) 2 mM Ca2+ + 1 mM Mg2+; or 4) 0.1 mM Mn2+. No attachment was observed in the absence of divalent cations, confirming that porcine trophectoderm attachment to SPP1 is integrin-mediated. This study is the first to demonstrate that a native form of uterine SPP1, secreted by the uterine glandular epithelium and present at the conceptus-maternal interface, can promote dose- and cation-dependent integrin-mediated adhesion of pig trophectoderm cells. (Supported by NRI USDA Grant 2006-35203-17199). (platform)

MUC1 is involved in trophoblast transendothelial migration

The factors that regulate trophoblast invasion of the uterine vasculature are incompletely understood. In this paper we show that macaque trophoblasts express the mucin, MUC1, and that it is involved in trophoblast-endothelial interaction. Immunocytochemistry, Western blotting and RT-PCR analyses confirmed that MUC1 was expressed by isolated early gestation macaque trophoblasts. MUC1 was also detected in endovascular trophoblasts in sections of placental–decidual tissue during early gestation. A blocking antibody against MUC1 reduced trophoblast adhesion to uterine endothelial cells and also blocked trophoblast transendothelial migration. MUC1 is known to bind to Intercellular Adhesion Molecule-1 (ICAM-1) in other systems. Incubation in the presence of a blocking antibody against Intercellular Adhesion Molecule-1 (ICAM-1) or recombinant ICAM-1 modestly, but significantly, reduced transendothelial trophoblast migration. These results are consistent with the idea that MUC1 is involved in trophoblast adhesion to uterine endothelial cells and in trophoblast transendothelial migration.

Trophoblast adhesion of the peri-implantation mouse blastocyst is regulated by integrin signaling that targets phospholipase C

Integrin signaling modulates trophoblast adhesion to extracellular matrices during blastocyst implantation. Fibronectin (FN)-binding activity on the apical surface of trophoblast cells is strengthened after elevation of intracellular Ca2+ downstream of integrin ligation by FN. We report here that phosphoinositide-specific phospholipase C (PLC) mediates Ca2+ signaling in response to FN. Pharmacological agents used to antagonize PLC (U73122) or the inositol phosphate receptor (Xestospongin C) inhibited FN-induced elevation of intracellular Ca2+ and prevented the upregulation of FN-binding activity. In contrast, inhibitors of Ca2+ influx through either voltage-gated or non-voltage-gated Ca2+ channels were without effect. Inhibition of protein tyrosine kinase activity by genistein, but not G-protein inhibition by suramin, blocked FN-induced intracellular Ca2+ signaling and upregulation of adhesion, consistent with involvement of PLC-γ. Confocal immunofluorescence imaging of peri-implantation blastocysts demonstrated that PLC-γ2, but not PLC-γ1 nor PLC-β1, accumulated near the outer surface of the embryo. Phosphotyrosine site-directed antibodies revealed phosphorylation of PLC-γ2, but not PLC-γ1, upon integrin ligation by FN. These data suggest that integrin-mediated activation of PLC-γ to initiate phosphoinositide signaling and intracellular Ca2+ mobilization is required for blastocyst adhesion to FN. Signaling cascades regulating PLC-γ could, therefore, control a critical feature of trophoblast differentiation during peri-implantation development.

Regulation of conceptus adhesion by endometrial CXC chemokines during the implantation period in sheep

To gain a better understanding of biochemical mechanisms of conceptus adhesion to the maternal endometrium in ruminant ungulates, the present study was performed to clarify roles of chemokines and extracellular matrix (ECM) components in the regulation of ovine blastocyst attachment to the endometrium. In addition to the chemokine, interferon-gamma inducible protein 10 kDa (IP-10, CXCL10), the chemokine receptor, CXCR3, also recognizes two other chemokines; monokine induced by IFN-gamma (MIG, CXCL9) and IFN-inducible T cell alpha chemoattractant (I-TAC, CXCL11). Similar to CXCL10, CXCL9, and CXCL11 were expressed in the uterus during the peri-implantation period, and CXCL9 mRNA expression was stimulated in endometrial explants from day 14 cyclic ewes by the addition of IFN-tau or IFN-gamma. Without ECM components, conceptus cell adhesion was low on day 14 of gestation and exhibited a 2.5-fold increase on day 17; adhesiveness on day 20 was 1/10 of that on day 14. Among various ECM components examined, trophoblast adhesion was greatest when fibronectin was used. Although day 14 conceptuses did not show much adhesive activity to fibronectin, day 17 trophoblast, and day 20 chorionic membrane exhibited 2.3-fold and 50-fold increase, respectively, which was enhanced by treatment with CXCL9 or CXCL10. These results indicate that through endometrial fibronectin and chemokines, ovine conceptus cells gain the ability to attach to the endometrium during pre-implantation period; however, elucidation of molecular mechanisms by which the conceptus acquires the adhesive ability during this time period awaits further investigation.