Oxygen regulates human cytotrophoblast migration by controlling chemokine and receptor expression.

INTRODUCTION: Placental development involves the variation of oxygen supply due to vascular changes and cytotrophoblast invasion. Chemokines and their receptors play an important role during placental formation. Herein, the analysis of the chemokine/receptor pair CXCL12/CXCR4 and further chemokine receptors, such as CCR1, CCR7 and CXCR6 expression in human cytotrophoblasts was conducted. METHODS: Human cytotrophoblasts were examined directly after isolation or after incubation with different oxygen tensions and a chemical HIF-stimulator for 12 h with realtime PCR, immunoblot, immunohistochemistry. Conditioned media of placental villi, decidua, and endothelial cells was used for ELISA analysis of CXL12. Cytotrophoblast migration assays were conducted applying conditioned media of endothelial cells, a CXCL12 gradient, and different oxygen level. Endometrial and decidual tissue was stained for CXCL12 expression. RESULTS: An upregulation of CXCL12, CXCR4, CCR1, CCR7 and CXCR6 was observed after cytotrophoblast differentiation. Low oxygen supply upregulated CXCR4, CCR7 and CXCR6, but downregulated CXCL12 and CCR1. In contrast to the HIF associated upregulation of the aforementioned proteins, downregulation of CXCL12 and CCR1 seemed to be HIF independent. Cytotrophoblast migration was stimulated by low oxygen, the application of a CXCL12 gradient and endothelial cell conditioned media. CXCL12 was detected in endometrial vessels, glands and conditioned media of placental and decidual tissue, but not decidual vessels. DISCUSSION/CONCLUSION: Taken together, oxygen supply and cytotrophoblast differentiation seem to be regulators of chemokine and receptor expression and function in human cytotrophoblasts. Therefore, this system seems to be involved in placental development, directed cytotrophoblast migration in the decidual compartment and a subsequent sufficient supply of the growing fetus.

Syndecan-1 knockdown in endometrial epithelial cells alters their apoptotic protein profile and enhances the inducibility of apoptosis.

Endometrial epithelial cells are known to undergo apoptosis during trophoblast invasion. We postulate that the cell surface molecule Syndecan-1 which is expressed on endometrial cells and syncytiotrophoblast is important for implantation in general and especially for induction of maternal cell apoptosis during trophoblast invasion because Syndecan-1's influence on apoptotic susceptibility of cancer cells is already described in the literature. Using the human endometrial epithelial cell line RL95-2, a new stable cell line with Syndecan-1 knockdown was generated. Via antibody array analysis, a significant decrease in the expression of anti-apoptotic proteins like inhibitors of apoptosis, Clusterin, heme oxygenase (HO-2), heat shock protein (HSP)27 and -70 and Survivin due to the Syndecan-1 knockdown was discovered. Correspondingly, active Caspase-3 as an indicator for apoptosis was increased more severely in these cells compared with unmodified RL95-2 after treatment with implantation-related stimuli, which are the cytokines interleukin-1ß, interferon-γ, tumor necrosis factor-α and transforming growth factor-ß1 and an anti-Fas antibody. Furthermore, a treatment with a combination of all factors caused a higher Caspase-3 induction compared with each single treatment. These results demonstrate that Syndecan-1 is involved in the control of apoptosis in RL95-2 cells and therefore may affect the fine tuning of apoptosis in endometrial epithelium regulating the embryo's invasion depth as a crucial step for regular implantation followed by successful pregnancy.

The human oviduct transcriptome reveals an anti-inflammatory, anti-angiogenic, secretory and matrix-stable environment during embryo transit.

The human oviduct serves as a conduit for spermatozoa in the peri-ovulatory phase and nurtures and facilitates transport of the developing embryo for nidation during the luteal phase. Interactions between the embryo and oviductal epithelial surface proteins and secreted products during embryo transit are largely undefined. This study investigated gene expression in the human oviduct in the early luteal versus follicular phases to identify candidate genes and biomolecular processes that may participate in maturation and transport of the embryo as it traverses this tissue. Oviductal RNA was hybridized to oligonucleotide arrays and resulting data were analysed by bioinformatic approaches. There were 650 genes significantly down-regulated and 683 genes significantly up-regulated (P<0.05) in the luteal versus follicular phase. Quantitative real-time PCR, immunoblot analysis and immunohistochemistry confirmed selected gene expression and cellular protein localization. Down-regulated genes involved macrophage recruitment, immunomodulation and matrix-degeneration, and up-regulated genes involved anti-inflammatory, ion transport, anti-angiogenic and early pregnancy recognition. The oviduct displayed some similarities and differences in progesterone-regulated genes compared with the human endometrium. Together, these data suggest a unique hormonally regulated environment during embryo development, maturation and transport through human oviduct and some conservation of progesterone signalling in tissues of common embryological origin. The oviduct serves as a conduit for spermatozoa in the peri-ovulatory phase and it nurtures and facilitates transport of the developing embryo during the luteal phase of the menstrual cycle, although precise interactions between the embryo and oviductal epithelium and secreted products are largely undefined. Herein, we investigated gene expression in human oviduct to identify candidate genes and processes that may participate in maturation and transport of the embryo as it develops implantation competence. Total RNA from human ampullary oviducts in the early luteal versus follicular phases was isolated and hybridized to oligonucleotide arrays. The data, analysed by bioinformatic approaches, revealed that 650 genes were significantly down- and 683 genes were significantly up-regulated in the luteal phase. Quantitative real-time PCR, immunoblot analysis and immunohistochemistry confirmed selected gene expression and cellular protein localization. The data demonstrated down-regulation of genes involved in macrophage recruitment, immunomodulation and matrix degeneration and up-regulation of ion transport and secretions, as well as anti-angiogenic and early pregnancy recognition. Together, these data suggest a unique hormonally regulated environment during embryo development, maturation and transport through the human oviduct and provide insight into mechanisms influencing acquisition of implantation competence of the human embryo during its passage through the oviduct en route to the uterine endometrium.

Selection of the in vitro culture media influences mRNA expression of Hedgehog genes, Il-6, and important genes regarding reactive oxygen species in single murine preimplantation embryos.

BACKGROUND: The aim of this paper was to determine the influence of different in vitro culture media on mRNA expression of Hedgehog genes, il-6, and important genes regarding reactive oxygen species in single mouse embryos. METHODS: Reverse transcription of single embryos either cultured in vitro from day 0.5 until 3.5 (COOK's Cleavage medium or Vitrolife's G-1 PLUS medium) or in vivo until day 3.5 post coitum. PCR was carried out for ß-actin followed by nested-PCR for shh, ihh, il-6, nox, gpx4, gpx1, and prdx2. RESULTS: The number of murine blastocysts cultured in COOK medium which expressed il-6, gpx4, gpx1, and prdx2 mRNA differed significantly compared to the in vivo group. Except for nox, the mRNA profile of the Vitrolife media group embryos varied significantly from the in vivo ones regarding the number of blastocysts expressing the mRNA of shh, ihh, il-6, gpx4, gpx1 and prdx2. CONCLUSIONS: The present study shows that different in vitro culture media lead to different mRNA expression profiles during early development. Even the newly developed in vitro culture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies.

CXCR7 and syndecan-4 are potential receptors for CXCL12 in human cytotrophoblasts.

The placenta forms the interface between the mother and the fetus. During placental development cytotrophoblasts differentiate to form the syncytium or to invade the decidual wall to breach maternal vessels and establish the blood flow in the intervillous space. This process is still not well understood but it is proposed that chemokines and their receptors are involved in guiding cytotrophoblasts to the decidua and maternal vessels as well as attracting immunocompetent cells to the implantation site. CXCL12 is a chemokine expressed by cytotrophoblasts and is involved in cytotrophoblast invasion, differentiation and survival. One of its receptors, CXCR4, has been detected on cytotrophoblasts. Recent data show that CXCR7 and syndecan-4 might partially mediate CXCL12 function in other cell types. In this study, we examined CXCR7 and syndecan-4 expression at the maternal-fetal interface via immmunolocalization in placental tissue sections and in isolated cytotrophoblasts. We further used immunoblot analyses to confirm the data. We were able to show that cytotrophoblasts express both receptors and that upregulation occurs during the differentiation process of cytotrophoblasts towards the invasive phenotype. On a functional level CXCR7 seems not to be involved in JAR cell chemotaxis, suggesting a different function of this receptor. In conclusion, we propose that CXCL12 binds to CXCR4, but also to CXCR7 and syndecan-4. These three receptors could mediate different functions of CXCL12, such as cell migration, directed invasion, proliferation and survival. The latter molecules might also be involved in the development of placental pathologies, such as preeclampsia or choriocarcinoma growth.

In vitro culture does not alter the expression of vascular endothelial growth factor and its receptors in single murine preimplantation embryos.

BACKGROUND: In vitro culture of embryos, as widely used in assisted reproduction techniques, may influence embryonic development and subsequently the establishment of pregnancy. The aim of this study was to determine a potential influence of the in vitroculture regarding VEGF, VEGFR1 and VEGFR2 mRNA expression in developing single mouse embryos. METHODS: Murine embryos were isolated on day 1 post coitus (p.c.) and cultivated for a developmental time course followed by examination for mRNA expression using RT-nested PCR. Furthermore, in vitro cultured blastocysts were compared to in vivo development at 101 h p.c. RESULTS: At 101 h p.c. there were no significant differences between in vivo and in vitro cultured blastocysts regarding the expression of VEGF and its receptors. In the developmental time course, VEGF expression increased up to 94% in late blastocysts whereas the VEGF receptor expression remained low. CONCLUSIONS: This study showed that the in vitro culture did not alter the embryonic VEGF and VEGFR mRNA expression reassuring that the culture conditions in assisted reproduction techniques are well suited for maintaining the VEGF mRNA expression profile. Additionally, nearly 100% VEGF expression in late blastocysts highlights its importance for angiogenesis induction at the fetal-maternal interface.

The embryo's cystatin C and F expression functions as a protective mechanism against the maternal proteinase cathepsin S in mice.

A successful implantation of a mammalian embryo into the maternal endometrium depends on a highly synchronized fetal-maternal dialogue involving chemokines, growth factors, and matrix-modifying enzymes. A growing body of evidence suggests an important role for proteinases playing a role in matrix degeneration and enhancing the embryo's invasive capacity and influencing the mother's immunological status in favor of the conceptus. This study focused on the expression of cathepsin S (CTSS) and its inhibitors in the murine fetal-maternal interface as well as the detection of the cellular sources of either proteinase and inhibitors. Nested RT-PCR for detection of embryonic mRNAs, immunohistochemistry of maternal and fetal tissues in B6C3F1 mice, and FACS analysis for determination of immunocompetent cell population were applied. This study shows that the cysteine proteinase CTSS is upregulated in the stroma of the implantation site, and that pregnancy induces an influx of CTSS-positive uterine natural killer cells. Compared to maternal tissues, the CTSS inhibitors cystatin F and C, but not the proteinase itself, are expressed in blastocysts. In conclusion, CTSS underlies a hormonal regulation in the maternal tissue and therewith most likely supports the embryonic implantation. The invading embryo regulates the depth of its own invasion through the expression of the cathepsin inhibitors and furthermore, interleukin-6 to activate CTSS in maternal tissues. Additionally, the observed decrease in CD3(+) cells leads to the hypothesis that cells of the cytotoxic T-cell group are down-regulated in the decidua to support the implantation and ensure the survival of the embryo.

Interleukin-1 system in the human fallopian tube-No spatial but a temporal regulation of mRNA and protein expression.

The human fallopian tube provides the environment for the first 5 days of embryonic development in vivo. The IL-1 system is involved in human embryo implantation. This study aimed to investigate IL-1beta, IL-1ra and IL-1R tI expression within the length of the human fallopian tube on mRNA- and protein-level in samples from proliferative versus secretory phase, postmenopause (PMP) samples and samples from intra- (IUP) and extrauterine pregnancies (EUP) to examine possible spatial and hormonal induced changes (fimbrial, ampullary and isthmic tube segments). On mRNA-level, IL-1beta was expressed in all samples except in PMP. IL-1R tI could be detected in all samples whereas IL-1ra was only expressed in secretory phase and the IUP sample. Immunohistochemically we could detect IL-1beta and IL-1R t1 protein in all proliferative and secretory phase samples with maximum intensity in secretory phase samples whereas IL-1ra was expressed in secretory phase samples only. Overall no spatial but temporal differences possibly due to hormonal changes could be observed suggesting a precise regulation of the IL-1 system, especially for IL-1ra and moreover a stable molecular architecture within the full length of the fallopian tube.

Expression of the vascular endothelial growth factor receptor neuropilin-1 in the human endometrium.

Angiogenesis is a key process in the endometrium which undergoes dramatic changes during the menstrual cycle. Molecules such as vascular endothelial growth factor (VEGF), acting via two tyrosine kinase family receptors (VEGFR1 [Flt-1] and VEGFR2 [KDR/Flk-1]), are potent modulators of angiogenesis and vascular remodelling in the endometrium. Recently, neuropilin-1 (NRP-1) was shown to be expressed in endothelial cells binding VEGF(165) and therewith enhancing the binding of VEGF(165) to VEGFR2. This suggests that NRP-1, in addition to the known VEGF receptors, may play an important role in VEGF-induced angiogenesis. In this study, the expression of NRP-1 in the cycling human endometrium has been investigated by reverse transcription (RT)-polymerase chain reaction (RT-PCR), semi-quantitative competitive RT-PCR (RT-cPCR) and immunohistochemical staining. NRP-1 was expressed in all 32 endometrium samples throughout the menstrual cycle. However, samples from the proliferative phase showed significantly higher expression levels of NRP-1 mRNA compared to samples from the secretory phase (t/c-ratio 2.13 vs. 0.84, p<0.05). Immunohistochemistry confirmed the results showing increased NRP-1 staining in vascular endothelium, glandular epithelium and stromal cells of the proliferative phase endometrium. This study demonstrates mRNA and protein expression of NRP-1 in human endometrium samples throughout the menstrual cycle. The enhanced expression of NRP-1 in the proliferative phase suggests that it may participate in hormonally regulated changes of endometrial angiogenesis, preparing the endometrium for the implantation of an embryo. NRP-1 expression might act as a co-factor for VEGF(165) enhancing the angiogenic stimulus.

Decidual stromal cell response to paracrine signals from the trophoblast: amplification of immune and angiogenic modulators.

During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast differentiation and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important for successful embryonic implantation, including establishing the placental vasculature, anchoring the placenta to the uterine wall, and promoting the immunoacceptance of the fetal allograph. To our knowledge, global crosstalk between the trophoblast and the decidua has not been elucidated to date, and the present study used a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and further treated with conditioned media from human trophoblasts (TCM) or, as a control, with control conditioned media (CCM) from nondecidualized stromal cells for 0, 3, and 12 h. Total RNA was isolated and processed for analysis on whole-genome, high-density oligonucleotide arrays containing 54,600 genes. We found that 1374 genes were significantly upregulated and that 3443 genes were significantly downregulated after 12 h of coincubation of stromal cells with TCM, compared to CCM. Among the most upregulated genes were the chemokines CXCL1 (GRO1) and IL8,CXCR4, and other genes involved in the immune response (CCL8 [SCYA8], pentraxin 3 (PTX3), IL6, and interferon-regulated and -related genes) as well as TNFAIP6 (tumor necrosis factor alpha-induced protein 6) and metalloproteinases (MMP1, MMP10, and MMP14). Among the downregulated genes were growth factors, e.g., IGF1, FGF1, TGFB1, and angiopoietin-1, and genes involved in Wnt signaling (WNT4 and FZD). Real-time RT-PCR and ELISAs, as well as immunohistochemical analysis of human placental bed specimens, confirmed these data for representative genes of both up- and downregulated groups. The data demonstrate a significant induction of proinflammatory cytokines and chemokines, as well as angiogenic/static factors in decidualized endometrial stromal cells in response to trophoblast-secreted products. The data suggest that the trophoblast acts to alter the local immune environment of the decidua to facilitate the process of implantation and ensure an enriched cytokine/chemokine environment while limiting the mitotic activity of the stromal cells during the invasive phase of implantation.

Angiopoietin-1 and -2 mRNA and protein expression in mouse preimplantation embryos and uteri suggests a role in angiogenesis during implantation.

After attachment and migration through the endometrial epithelium, the embryo must induce angiogenesis within the endometrial stroma to successfully complete the implantation process. Growth factors have been shown to play an important role in embryo implantation and placentation. The aim of the study was to investigate the expression of angiopoietin-1 and -2 (Ang-1 and -2) mRNA and protein expression during the development of single preimplantation mouse embryos and of possible complementary expression in mouse uteri. Angiopoietin-1 mRNA was expressed throughout development in 78% of zygotes, 66% of 2-cell-embryos, 71% of 4-cell-embryos, 70% of 8-cell-embryos, 60% of morula stages, 48% of early blastocysts and 78% of late blastocysts. The number of Ang-1-expressing embryos in the early-blastocyst group was significantly different in comparison with zygotes, 4-cell-embryos, 8-cell-embryos and late blastocysts. Angiopoietin-2 mRNA and protein expression could not be detected in preimplantation embryos. Examination of the uteri revealed Ang-2 mRNA and protein expression in the oestrogen-dominated cycling phase and the progesterone-dominated mated phase, whereas Ang-1 expression was restricted to the mated phase. Herein, Ang-1 expression in preimplantation mouse embryos as well as Ang-1 and -2 expression in mouse uteri is demonstrated, suggesting a possible role for angiopoietins in the embryo-maternal dialogue of the implantation process via an enhancement of the vascular remodelling in favour of an implanting conceptus.

Dose-dependent insulin regulation of insulin-like growth factor binding protein-1 in human endometrial stromal cells is mediated by distinct signaling pathways.

IGF binding protein-1 (IGFBP-1) is a major product of decidualized human endometrial stromal cells and decidua, and as a modulator of IGF action and/or by independent mechanisms, it regulates cell growth and differentiation and embryonic implantation in these tissues. IGFBP-1 secretion is primarily stimulated by progesterone and cAMP and is inhibited by insulin and IGFs. The signaling pathways mediating the latter are not well defined, and the current study was conducted to determine which pathways mediate the effects of insulin on IGFBP-1 mRNA and protein expression by human endometrial stromal cells decidualized in vitro by progesterone. Cells were cultured and treated with different combinations of insulin; wortmannin, an inhibitor of the phosphatidylinositide-3-kinase (PI3-kinase) pathway; and PD98059, an inhibitor of the MAPK pathway. IGFBP-1 mRNA was determined by real-time PCR, and protein secretion in the conditioned medium was measured by ELISA. Activation of the PI3-kinase and the MAPK pathways was assessed by the detection of phosphorylated AKT and ERK in Western blots, respectively. Insulin inhibited IGFBP-1 mRNA and protein secretion in a dose-dependent fashion, with an ED(50) for the latter 0.127 ng/ml (21.6 pm). Inhibitor studies revealed that at low doses, insulin acts through the PI3-kinase pathway, whereas at higher levels it also activates the MAPK pathway in the inhibition of IGFBP-1. The data demonstrate that human endometrium is a target for insulin action in the regulation of IGFBP-1. At physiological levels insulin likely plays a homeostatic role for energy metabolism in the endometrium, and in hyperinsulinemic states, insulin action on the endometrium may activate cellular mitosis via the MAPK pathway and perhaps predispose this tissue to hyperplasia and/or cancer.