Expression and regulation of vascular endothelial growth factor in a first trimester trophoblast cell line.

Embryo implantation and development are critically dependent upon the spatial and temporal regulation of angiogenesis and localized vascular permeability. A key mediator of these effects is the endothelial cell mitogen vascular endothelial growth factor (VEGF). VEGF has been shown to promote endometrial vascular permeability, fetal vasculogenesis and placental, fetal and maternal angiogenesis. However, the mechanism through which this regulation occurs in the placenta is poorly understood. This study was conducted to determine if the pro-angiogenic cytokines, TNF-alpha and TGF-beta1, affect VEGF expression in human first trimester trophoblasts. Culture of a first trimester trophoblast cell line (HTR-8/SVneo), in the presence of either TNF-alpha or TGF-beta1, resulted in the expression of significant levels of VEGF in culture. The trophoblast cell line also showed a time-dependent and a dose-dependent increase in VEGF mRNA levels when cultured in the presence of either TNF-alpha or TGF-beta1. These results suggest that both TNF-alpha and TGF-beta1 may regulate the production of VEGF in early gestational trophoblasts and may therefore serve to modulate placental vascular permeability and angiogenesis that are necessary for embryo implantation and placentation.

Adrenomedullin levels in normal and preeclamptic pregnancy at term.

OBJECTIVE: To describe maternal plasma levels of adrenomedullin (AM), a hypotensive and natriuretic peptide, in normal and preeclamptic women at term. STUDY DESIGN: Maternal plasma AM levels were determined in 13 preeclamptic and 15 normotensive primigravidas by radioimmunoassay. Plasma samples were obtained with the patients in the lateral recumbent position before the administration of any medications. RESULTS: Women with preeclampsia had significantly elevated AM levels when compared with normotensive controls (42.3 +/- 10.5 pg/mL versus 16.9 +/- 3.1 pg/mL, P < .011). CONCLUSION: In this pilot study, AM levels were significantly increased at term in preeclamptic women.

Ethanol-induced expression of cytokines in a first-trimester trophoblast cell line.

OBJECTIVES: Altered cytokine expression at the fetoplacental interface may be a potential mechanism for the development of fetal immune dysfunction in children with fetal alcohol syndrome. This study was conducted to determine whether first-trimester trophoblasts respond to ethanol exposure by the induction of specific cytokines. STUDY DESIGN: HTR-8/SVneo trophoblast cells were cultured in vitro in the presence of either ethanol (0.5% [vol/vol]), lipopolysaccharide (1 microg/mL), or ethanol and lipopolysaccharide. Expression of granulocyte colony-stimulating factor, regulated on activation normal T cell expressed and secreted, and interleukin-6 was examined by Northern analysis and enzyme-linked immunosorbent assay. RESULTS: Culture in the presence of ethanol, lipopolysaccharide, or lipopolysaccharide and ethanol resulted in the increased transcription and secretion of granulocyte colony-stimulating factor, regulated on activation normal T cell expressed and secreted, and interleukin-6 at significantly greater levels (P < .01) than control cultures. CONCLUSIONS: Human first-trimester trophoblasts express high levels of cytokines when cultured in the presence of ethanol. Trophoblasts may therefore be an important exogenous source of cytokines for the fetus, and altered cytokine levels during early gestation may have an adverse effect on the development of the fetal immune system.

Effects of tumor necrosis factor-alpha on human trophoblast cell adhesion and motility.

PROBLEM: Adhesive interaction between trophoblast cells and uterine endometrial basement membrane is one of the critical processes in embryo implantation. This interaction is directly or indirectly regulated by hormones, growth factors, and cytokines. Since tumor necrosis factor-alpha (TNF-alpha) is synthesized by both decidual and trophoblast cells, we hypothesized that TNF-alpha may play a regulatory role in trophoblast cell invasion. To test this hypothesis, we have used in vitro models to determine the effect of TNF-alpha on human trophoblast cell adhesion and motility, two major steps in trophoblast invasion. METHODS: The effect of TNF-alpha on the motility of extended-lifespan first trimester trophoblasts (HTR) and JEG-3 choriocarcinoma cells was tested using the phagokinetic track motility assay. An in vitro adhesion assay was used to determine the effect of TNF-alpha on the adhesion of HTR and JEG-3 cells to laminin, a major basement membrane component. In addition, the effect of TNF-alpha on the surface expression of the laminin receptor beta 1 integrin subunit was examined using flow cytometry. RESULTS: HTR or JEG-3 cells strongly adherent to laminin which was not significantly altered by TNF-alpha treatment. We also measured the effect of TNF-alpha on the surface expression of beta 1 integrin on HTR and JEG-3 cells; no difference was observed between control and treatment groups. Interestingly, the motility of both HTR and choriocarcinoma JEG-3 cells was significantly inhibited by TNF-alpha. CONCLUSIONS: The role of TNF-alpha in human embryo implantation is currently unknown. Our data demonstrate that TNF-alpha does alter trophoblast cell adhesion to laminin, but significantly inhibits trophoblast cell motility in vitro, suggesting that TNF-alpha may play a regulatory role in trophoblast cell invasion.

Expression and function of autocrine motility factor receptor in human choriocarcinoma.

OBJECTIVE: Our purpose was to determine whether human choriocarcinoma cells express autocrine motility factor receptor (AMF-R) and to study its function in this tumor system. STUDY DESIGN: The expression and localization of AMF-R were compared in choriocarcinoma and normal placental trophoblasts using both cell lines and tissue sections. In addition, migratory properties of choriocarcinoma cells and normal placental cells was determined. RESULTS: Using immunofluorescence and immunoperoxidase staining, we have detected the expression of AMF-R in choriocarcinoma cells with receptor clustering on the cell surface, while term placenta cells expressed AMF-R less intensely with no receptor clustering. In choriocarcinoma tissues, AMF-R was strongly expressed in malignant cytotrophoblasts cells while adjacent normal villous trophoblast cells and necrotic regions were weakly or negatively stained. Choriocarcinoma cells responded to AMF-R stimulation with increased cell motility, while term placental cells were unresponsive. CONCLUSION: Human choriocarcinoma cells express functional cell surface AMF-R in vitro and in choriocarcinoma tissue suggesting that this receptor may play an important role in cancer cell motility.

Human trophoblast cell adhesion to extracellular matrix protein, entactin.

PROBLEM: Trophoblast interaction with endometrial extracellular matrix (ECM) is crucial during human embryo implantation and placentation. Entactin, a ubiquitous basement membrane glycoprotein, plays a central role in ECM assembly, cell attachment, and chemotaxis. The present study was conducted to examine the possible role of entactin in promoting human trophoblast adhesion. METHODS: Using an extended life span first trimester trophoblast cell line HTR-8/SVneo (HTR) and a cell adhesion assay, we measured the adherence of human first trimester trophoblasts to recombinant entactin and its domains. Also, we used flow cytometry and indirect immunofluorescence to detect the presence of integrins that may be involved in human trophoblast-entactin interaction; these methods were used to analyze HTR cells, as well as tissue sections and freshly isolated human trophoblasts from first trimester and term placenta. RESULTS: We found that first trimester trophoblast cells were highly adherent to entactin and its E and G2 domains but not to G1 or G3 domains. Using indirect immunofluorescence and flow cytometry, we found that both beta 1 and beta 3 integrin subunits were expressed on the surface of HTR trophoblast cells adhering to entactin; in contrast, beta 2 and beta 4 integrin subunits were not detected. In addition, we found that alpha v beta 3 was expressed on freshly isolated villous cytotrophoblasts and cytotrophoblast and syncytiotrophoblasts in tissue sections from term placenta. The beta 3 integrin subunit was expressed in cytotrophoblasts and syncytiotrophoblasts in villi of first trimester placental tissue sections. CONCLUSION: Recombinant entactin promotes human trophoblast cell adhesion through both its E and G2 domains and these specific adhesive interactions may be mediated by beta 1 and/or beta 3 class integrins.

Brain natriuretic peptide and cyclic guanosine-3',5' monophosphate in pre-eclampsia.

The objective of this study is to determine the possibility that pre-eclampsia, a disease characterized by altered vascular tone, may result in altered levels of fetal BNP and cGMP, and to determine whether pre-eclampsia alters the maternal-fetal relationship of BNP and cGMP. Paired maternal and umbilical venous plasma levels of BNP and cGMP were determined in 13 pre-eclamptic and 9 normotensive primigravidas in the third trimester. Statistical analysis was performed using multivariate analysis of variance, linear regression, and canonical correlation. Overall, levels of cGMP were lower in pre-eclampsia (P < 0.03). Pre-eclampsia was also associated with an altered maternal-fetal relationship for BNP and cGMP (P < 0.008, P < 0.02, respectively). With pre-eclampsia, the maternal:fetal ratio was reduced for BNP and was increased for cGMP. Because of its role as a second messenger for many vasoactive hormones, we hypothesize that fetal cGMP levels may better reflect overall vascular tone than do individual hormones. Altered BNP and cGMP maternal-fetal homeostasis raises the possibility of maternal-fetal coordination of vascular control.

Molecular interactions between fibronectin and integrins during mouse blastocyst outgrowth.

To investigate the mechanism of trophoblast adhesion to fibronectin, we cultured blastocysts in serum-free medium on proteolytic fibronectin fragments containing its major functional domains, and localized fibronectin-binding integrins in outgrowing trophoblast cells by immunofluorescent staining. Outgrowth comparable to that obtained with intact fibronectin was observed using a 120 kD chymotryptic fragment containing the central cell-binding domain (FN-120) and the Arg-Gly-Asp (RGD) recognition sequence. A 40 kD COOH-terminal chymotryptic fragment of fibronectin containing both a heparin-binding region and an alternate (non-RGD) cell-binding site was inactive in supporting trophoblast adhesion. Three synthetic peptides derived from the heparin-binding domain, including the CS1 alternate cell-binding site, were also unable to promote trophoblast cell adhesion. A 75 kD recombinant protein, ProNectin F, containing 13 copies of the cell recognition epitope of fibronectin, Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser, vigorously supported blastocyst outgrowth. Blastocyst outgrowth was not significantly different when surfaces were precoated with cellular fibronectin, which contains an alternatively spliced type III repeat and is the form actually encountered in vivo. Several putative fibronectin receptors were localized in trophoblast outgrowths by immunofluorescent labeling. Antibodies reactive with integrin subunits alpha 3, alpha 5, alpha IIb, alpha v, beta 1 and beta 3, but not alpha 4, all bound to trophoblast cells. Antibodies raised against either the beta 1 or beta 3 integrin subunits significantly inhibited fibronectin-mediated outgrowth. These findings demonstrate the key role of the central cell-binding domain of fibronectin in trophoblast adhesion, and suggest four RGD-binding integrins, alpha 3 beta 1, alpha 5 beta 1, alpha IIb beta 3, and alpha v beta 3, that could mediate trophoblast adhesion in vitro and may play an important role during implantation.

Ultrastructural and immunocytochemical studies of iris vessels in rats with experimental autoimmune uveoretinitis.

In this study, we describe the ultrastructural and immunocytochemical changes that occur in the iris of rats with experimental autoimmune uveoretinitis (EAU). General changes include perivascular infiltration of inflammatory cells, followed by hemorrhage and extensive tissue destruction. Alterations in the iris epithelium were also noted. A breakdown in the blood-iris barrier was demonstrated in some vessels at the peak of inflammation; peroxidase reaction product was seen in the basement membrane and perivascular spaces. We found that, in inflamed iris vessels, endothelial cells and smooth muscle cells become hypertrophic and show increased amounts of synthetic organelles. This finding is similar to our previous observations on endothelial cells and smooth muscle cells in retinal vessels in EAU. In addition, as was reported in the retinal vascular basement membrane in EAU, there is an increase in immunoreactivity of several extracellular matrix molecules in the iris vascular basement membrane; during inflammation, there is a significant increase in immunoreactivity of collagen types I and III, entactin, fibronectin, and laminin. Activated endothelial cells and smooth muscle cells are likely to be involved in the synthesis of certain of these matrix molecules.

Mouse blastocyst outgrowth and implantation rates following exposure to ethanol or A23187 during culture in vitro.

This study focused on the effects of ethanol on blastocyst outgrowth and implantation in mice. Blastocysts were exposed to ethanol in Ham's F10 medium and then cultured free of ethanol on fibronectin-coated Petri dishes to assess trophoblast cell adhesion and migration. The time necessary for half of the embryos to outgrow was significantly less (P < 0.05) following treatment with 0.1%, 0.2%, 0.4% or 1.0% (w/v) ethanol for either 5 min or 24 h compared with controls. The rate of trophoblast cell migration was determined by measuring the mean area of outgrowing embryos using an image analysis system. Blastocysts exposed to ethanol for 5 min produced a greater (P < 0.05) average outgrowth area than did stage-matched controls. Acceleration of blastocyst cavitation by ethanol is known to be associated with an increase in the intracellular concentration of calcium. Here, treatment with the calcium ionophore A23187 stimulated (P < 0.05) trophoblast outgrowth and accelerated (P < 0.05) the rate of cell migration. In an attempt to correlate the effect of ethanol on outgrowth in vitro with implantation in utero, cultured blastocysts were either not exposed to ethanol or exposed to 0.1% ethanol for 5 min and transferred 24 h later to the uteri of pseudopregnant dams. The implantation rate (39.4%, n = 376) and the rate of development to term (2.45 pups per mouse, n = 20) were higher (P < 0.05) in mice receiving ethanol-treated embryos compared with those receiving control embryos (20.8%, n = 331; 1.16 pups per mouse, n = 18, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of in vitro cocaine exposure on human sperm motility, intracellular calcium, and oocyte penetration.

OBJECTIVE: To determine if cocaine exposure affects human sperm motility, intracellular calcium level, and fertilizing capability. DESIGN AND METHODS: Human semen samples were treated with 1 to 1,000 microM cocaine hydrochloride for up to 2 hours in vitro. Sperm motion kinematics were measured by computer-assisted semen analysis (CASA). Spermatozoan intracellular calcium was determined by laser cytometry. The sperm fertilizing capability was assessed using the zona-free hamster oocyte penetration test. RESULTS: After a short exposure (15 minutes) to cocaine, the sperm motion kinematic parameters, straight line velocity and linearity, were decreased in the high concentration groups. However, after a longer exposure (2 hours) to cocaine, the differences were no longer significant. Cocaine treatment did not alter spermatozoa intracellular calcium levels. Most importantly, human sperm treated with cocaine at a high concentration were fully capable of penetrating zona-free hamster oocytes. CONCLUSION: Human spermatozoa acutely exposed to high concentrations of cocaine initially demonstrate a decrease in two motion kinematics, straight line velocity and linearity. However, overall, cocaine exposure had no significant effects on sperm motility and fertilizing capability.

Blastocyst cavitation is accelerated by ethanol- or ionophore-induced elevation of intracellular calcium.

To extend our previous finding that ethanol stimulates murine preimplantation development, we focused in the current study on the cavitation and expansion of the blastocyst. Ethanol stimulation of blastocyst cavitation and expansion was optimal at a concentration of 0.1% and required only a 5-min exposure. Because intracellular levels of calcium were transiently increased in the ethanol-exposed embryos, we determined the effect of directly increasing calcium on the rates of blastocyst cavitation and expansion. Treatment with the calcium ionophore, A23187, altered development much as did ethanol, indicating that ethanol may stimulate preimplantation development by increasing the intracellular calcium concentration. The relationship between intracellular calcium levels and blastocoel volume suggests that morphogenetic events during preimplantation development, particularly cavitation and blastocyst formation, may be regulated through modulation of intracellular calcium levels.

Recombinant entactin promotes mouse primary trophoblast cell adhesion and migration through the Arg-Gly-Asp (RGD) recognition sequence.

In vitro culture of mouse blastocysts during the period coinciding with implantation has revealed that primary trophoblast cells can adhere and migrate in serum-free medium when provided with certain extracellular matrix components, including fibronectin and laminin. Tightly associated with laminin is the glycoprotein, entactin, that may play an important role in basement membrane assembly and cell attachment. Mouse blastocysts were studied using this in vitro model to determine whether entactin was capable of mediating trophoblast invasive activity. Although entactin has never been shown to promote cell migration, we report here that recombinant entactin supported blastocyst outgrowth in a dose-dependent manner, with a maximal effect at 20-50 micrograms/ml. The ability of trophoblast cells to adhere and migrate on entactin was specifically inhibited by anti-entactin antibody, but not by antibodies raised against laminin. The synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, that contains the Arg-Gly-Asp (RGD) integrin recognition site, reversibly inhibited entactin-mediated blastocyst outgrowth in a dose-dependent manner, but had no effect on laminin-mediated outgrowth. The synthetic peptide, Gly-Phe-Arg-Gly-Asp-Gly-Gln, that comprises the actual RGD-containing sequence within entactin, promoted trophoblast outgrowth when immobilized on the substratum. Furthermore, a mutated recombinant entactin, altered to contain a Glu in place of Asp at the RGD site, provided no trophoblast cell adhesive activity. We conclude that entactin promotes trophoblast outgrowth through a mechanism mediated by the RGD recognition site, and that it may play an important role during invasion of the endometrial basement membrane at implantation.

Effects of a DNA-specific fluorochrome, Hoechst 33258, on mouse sperm motility and fertilizing capacity.

DNA specific fluorochrome (Hoechst 33342 and 33258) as non-toxic stains, have been widely used to measure cell density and proliferation, detect sperm-egg fusion, and observe the development of pre-implantation embryos. It has been reported that Hoechst 33342 at a concentration of 10 micrograms ml-1 had significant inhibition on embryo cleavage. In this study, we incubated B6D2F1 mouse sperm and eggs with different concentrations of Hoechst 33258, 0, 1.0, 10.0, 20.0, 100 micrograms ml-1. We found that: (1) 100 micrograms ml-1 of H-33258 significantly decreased the sperm motility at 90 min and 4 h. (P less than 0.05), (2) 20 micrograms ml-1 and 100 micrograms ml-1 of Hoechst 33258 significantly inhibited mouse fertilization in vitro (P less than 0.05), and (3) 1.0 micrograms ml-1 and 10.0 micrograms ml-1 Hoechst 33258 had no effect on fertilization rate. But when we pre-incubated sperm at 10 micrograms ml-1 Hoechst 33258 for 90 min, and inseminated oocytes in the medium with same concentration of Hoechst 33258, the embryo cleavage was significantly inhibited.

Cellular toxicity of toluene on mouse gamete cells and preimplantation embryos.

Toluene is an aromatic hydrocarbon, which has been used in the paint, lacquer and glue industry. It has been detected in municipal water supplies. Previous mouse in vivo studies indicated that toluene administrated by gavage increased the embryonic mortality. The present in vitro study demonstrated that a concentration of toluene higher than 8.67 micrograms/ml not only decreased sperm motility and inhibited fertilization, but also significantly increased preimplantation embryo degeneration. At lower levels no effects were observed and the adverse effect levels were approximately 780 fold higher than reported levels in municipal water supplies.