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J D Aplin

Publications and source records attributed to J D Aplin.

At least 19 recordsLinked to original sources
PubMed2006

Dimethylarginine dimethylaminohydrolase (DDAH) regulates trophoblast invasion and motility through effects on nitric oxide.

BACKGROUND: Invasion of trophoblast into the uterine environment is crucial for establishing a successful pregnancy. Physiological production of nitric oxide (NO) by extravillous trophoblasts results in significant pro-invasive effects. NO synthesis is competitively inhibited by methylated arginine analogues such as asymmetric dimethylarginine (ADMA) but not the enantiomer symmetric dimethylarginine (SDMA). Within cells, the concentration of ADMA is regulated by the activity of the enzyme dimethylarginine dimethylaminohydrolase (DDAH). The aim of this study was to examine DDAH expression and function in trophoblasts. METHODS AND RESULTS: DDAH-1 and DDAH-2 messenger RNA and protein were demonstrated in first trimester placental tissue, primary extravillous trophoblasts and extravillous trophoblast-derived cell lines. DDAH activity was demonstrated in both cells and tissue. Overexpression of DDAH-1 in trophoblasts led to a number of significant changes, including an 8-fold increase in enzymatic activity, a 59% decrease in production of ADMA (but not SDMA), a 1.9-fold increase in NO and a 1.6-fold increase in cyclic guanosine monophosphate (cGMP) production. Functional assays showed that increased DDAH activity led to significantly increased cell motility and invasion in response to hepatocyte growth factor (HGF). CONCLUSIONS: DDAH may play an important role in the regulation of extravillous trophoblast function via its effects on ADMA and NO production.

Amidohydrolases↗
PubMed2006

Implantation of the human embryo: research lines and models. From the implantation research network 'Fruitful'.

Infertility is an increasing problem all over the world, and it has been estimated that 10-15% of couples in fertile age have fertility problems. Likewise induced unsafe abortion is a serious threat to women's health. Despite advances made in assisted reproduction techniques, little progress has been made in increasing the success rate during fertility treatment. This document describes a wide range of projects carried out to increase the understanding in the field of embryo implantation research. The 'Fruitful' research network was created to encourage collaborations within the consortium and to describe our different research potentials to granting agencies or private sponsors.

Animals↗
PubMed2006

Trophoblast differentiation: progenitor cells, fusion and migration -- a workshop report.

Challenge lies ahead in unravelling the role played by trophoblast and its repertoire of expressed genes in normal human placental development, growth and pathology. Specific technical advances will clearly be required for characterisation of function. In particular, improvements in our repertoire of in vitro models are needed before many of the key questions can be answered. Recent advances in the study of human trophoblast differentiation are discussed.

Cell Communication↗
PubMed2006

Embryo implantation: the molecular mechanism remains elusive.

Low rates of implantation are an impediment to more efficient assisted reproduction techniques. Improved endometrial receptivity and embryo preparation should lead to higher pregnancy rates, lower rates of early pregnancy failure and fewer multiple pregnancies. As the first site of contact between embryo and endometrium, the luminal epithelium (LE) is responsible for the non-receptive status of proliferative and early secretory tissue, and transformation to receptivity in the mid-secretory phase presumably requires alterations in expression, organization or activation of adhesion systems. Luminal cells are less abundant than their glandular counterparts, and are under-represented in global tissue datasets. Furthermore, alterations in cell surface composition can be readily accomplished by mechanisms that do not rely on altered transcription or translation. Current data from in-vitro models are consistent with initial attachment to mucin in the apical glycocalyx, perhaps via a carbohydrate-mediated interaction, after which the epithelial phenotype is modified by a medium- or short-range embryonic signal. A cascade of interactions follows, mediating embryo migration across the epithelium. Strikingly, numerous potential mediators of adhesion at implantation are located in the lateral rather than the apical surface of LE cells. Attached embryos appear to gain rapid access to this highly adhesive lateral membrane domain.

Adhesiveness↗
PubMed2005

Human placental explants in culture: approaches and assessments.

Placental explant cultures in vitro are useful for studying tissue functions including cellular uptake, production and release of secretory components, cell interactions, proliferation, growth and differentiation, gene delivery, pharmacology, toxicology, and disease processes. A variety of culture conditions are required to mimic in utero environments at different times of gestation including differing oxygen partial pressures, extracellular matrices and culture medium. Optimization of explant methods is examined for first and third trimester human placental tissue and the biological processes under investigation.

Biocompatible Materials↗
PubMed2004

Expression pattern of collagen XVIII and its cleavage product, the angiogenesis inhibitor endostatin, at the fetal-maternal interface.

Endostatin, the C-terminal fragment of the basement membrane protein collagen XVIII regulates epithelial cell migration and impairs tumour growth by inhibiting angiogenesis. Here, we investigated the expression pattern of collagen XVIII/endostatin in human placental and decidual tissues of various ages of gestation as well as in primary villous cytotrophoblasts, trophoblast cell lines, and villous explant cultures differentiating along the invasive pathway. RT-PCR analysis revealed production of collagen XVIII mRNA in total placenta and decidua of early and late pregnancy and in SGHPL-5 and HTR-8/Svneo cells. Collagen XVIII transcripts were absent from purified extravillous trophoblasts and syncytialising trophoblast cultures. Accordingly, an antibody against a protein domain common to different collagen XVIII isoforms detected the 180 kDa protein in villous and decidual tissue and cultivated placental fibroblasts but not in the different isolated trophoblast cell types. Immunohistochemical analyses localised collagen XVIII to villous basement membranes and to the endothelium as well as to placental and decidual stromal cells. Interestingly, expression of various forms of endostatin (20 and 26 kDa) was detected in placenta and decidua using Western blot analyses. Moreover, supplementation of recombinant endostatin increased MMP-2 expression in villous explant cultures and SGHPL-5 cells suggesting that the inhibitor may modulate extravillous trophoblast differentiation.

Adult↗
PubMed2003

Placental extracellular matrix: gene expression, deposition by placental fibroblasts and the effect of oxygen.

Database mining revealed 102 extracellular matrix (ECM) genes amongst about 10000 mRNA species expressed in human placenta, and these were classified into collagens (23), non-collagenous glycoproteins (59) and proteoglycans (23). A panel of antibodies to selected collagens and glycoproteins was used to examine ECM distribution in the placental villous stroma. Collagens I and IV, fibronectin and fibrillin I were abundant in first trimester and term tissue. Some areas lacked collagen I, while collagen IV was clearly evident in interstitial locations. At term, laminin was present in the stroma as well as in trophoblastic and vascular basement membranes. Thrombospondin I, tenascin C and elastin showed more restricted distributions. Fibrosis has been reported in association with ischaemia, so ECM production by cultured term and first trimester placental fibroblasts was evaluated at three different oxygen concentrations. Fibronectin and collagen IV were more strongly expressed than collagen I, fibrillin I or thrombospondin I, while the production of laminin and elastin was very low. Reducing the oxygen tension led to a selective increase in fibronectin and collagen IV production. Thus both quantitative and qualitative alterations in ECM composition may be expected to accompany prolonged hypoxia.

Adult↗
PubMed2003

Small leucine-rich proteoglycans (SLRPs) in uterine tissues during pregnancy in mice.

Remodelling of the extracellular matrix (ECM) occurs during decidualization of the endometrium in mice. Previously we have documented the appearance of large-diameter collagen fibrils around mature decidual cells between day 5 and day 7 of pregnancy. Proteoglycans are important in the regulation of collagen fibrillogenesis, and the present study analysed four members (decorin, biglycan, lumican and fibromodulin) of the family of small leucine-rich proteoglycans (SLRPs) in the uterus from day 1 to day 7 of pregnancy. Decorin was present together with lesser amounts of lumican in the stroma before the onset of decidualization, whereas biglycan and fibromodulin were almost absent. Biglycan and, less significantly, lumican were expressed in decidualized regions of the endometrium, but decorin was absent. Fibromodulin was weakly expressed in the non-decidualized stroma, but only after implantation. Decorin and lumican were strongly expressed in the undifferentiated interimplantation site stroma, whereas biglycan and fibromodulin were expressed only weakly. These results indicate that the SLRP profile of the uterine ECM alters with differentiation of endometrial stromal cells. The large decidual collagen fibrils are thought to arise by lateral association of smaller diameter fibrils. As decorin has been shown to inhibit lateral association of collagen fibrils, its disappearance between day 2 and day 5 of pregnancy may be a prerequisite for the formation of large fibrils in decidua in mice.

Animals↗
PubMed2001

alpha 2-Macroglobulin: a new component in the insulin-like growth factor/insulin-like growth factor binding protein-1 axis.

Insulin-like growth factors (IGFs) are crucial for many aspects of development, growth, and metabolism yet control of their activity by IGF-binding proteins (IGFBPs) remains controversial. The effect of IGFBP-1 depends on its phosphorylation status; phosphorylated IGFBP-1 inhibits IGF actions whereas the nonphosphorylated isoform is stimulatory. In order to understand this phenomenon, we purified phosphorylated IGFBP-1 from normal human plasma by immunoaffinity chromatography. Unexpectedly, the resulting preparation enhanced IGF-stimulated 3T3-L1 fibroblast proliferation, due to the presence of a co-purified protein of approximately 700 kDa. Matrix-assisted laser desorption ionization-mass spectrometry and Western immunoblotting analysis identified this co-purified protein as alpha(2)-macroglobulin (alpha(2)M). Anti-alpha(2)M antibodies co-immunoprecipitated IGFBP-1 from human plasma and from (125)I-IGFBP-1.alpha(2)M complexes formed in vitro. The (125)I-IGFBP-1/alpha(2)M association could be inhibited with excess unlabeled IGFBP-1. Surface plasmon resonance analysis indicated that alpha(2)M preferentially associates with the phosphorylated isoform of IGFBP-1 and that when complexed to alpha(2)M, IGFBP-1 can still bind IGF-I. These findings have functional significance since alpha(2)M protects IGFBP-1 from proteolysis and abrogates the inhibitory effect of phosphorylated IGFBP-1 on IGF-I stimulated 3T3-L1 cell proliferation. We conclude that alpha(2)M is a binding protein of IGFBP-1 which modifies IGF-I/IGFBP-1 actions resulting in enhanced IGF effects. In line with its role in regulating the clearance and activity of other growth factors, we predict that alpha(2)M has a novel and important role in controlling the transport and biological activity of IGFs.

3T3 Cells↗
PubMed2001

MUC1, glycans and the cell-surface barrier to embryo implantation.

As it approaches the maternal surface, the attaching embryo encounters the epithelial glycocalyx, which contains the mucin, MUC1. A high density of MUC1 at the cell surface can inhibit cell adhesion. This raises the possibility of the existence of a uterine barrier to implantation that might allow maternal rejection of poorer quality embryos. To investigate the mechanism of implantation, human embryos were incubated with endometrial epithelial monolayers. Hatched blastocysts were found to attach readily to the epithelial surface. MUC1 was lost from epithelial cells beneath and near to the attached embryo, while normal expression persisted in neighbouring cells.

Animals↗
PubMed2001

Regulation of IGF bioavailability in pregnancy.

During pregnancy, insulin-like growth factors (IGFs) are important for growth of fetal and maternal tissues. One of the IGF binding proteins, IGFBP-1, is thought to regulate their activity within the local environment of the placenta. IGFBP-1 usually exists as a phosphorylated, high affinity species, which sequesters IGFs, thereby inhibiting their actions. This study has investigated the mechanisms that release IGF from IGFBP-1 at the maternal-fetal interface. Under basal conditions, human decidualized endometrium produces both non-phosphorylated (np) and phosphorylated (p) isoforms of IGFBP-1; however, in the presence of IGF-II, which is a trophoblast secretory product, npIGFBP-1 was preferentially produced. Furthermore, we found that trophoblast, presumably via placental alkaline phosphatase, can de-phosphorylate pIGFBP-1. Since npIGFBP-1 has decreased affinity for IGF-I, these effects should enhance IGF-I bioavailability. In addition, we found that decidual cells produce a protease, which cleaves IGFBP-1, but only when it is non-phosphorylated; [(125)I]-npIGFBP-1 is proteolysed into 14 and 17 kDa fragments which have markedly reduced affinity for IGF. We therefore propose paracrine modulation of IGFBP-1 at the maternal-fetal interface involving a multi-step process of de-phosphorylation and proteolysis; this will result in enhanced IGF bioavailability and is likely to represent an important mechanism for controlling fetal and maternal tissue growth.

Biological Availability↗
PubMed2001

Human endometrial mucin MUC1 is up-regulated by progesterone and down-regulated in vitro by the human blastocyst.

Expression of MUC1 in endometrial epithelium has been suggested to create a barrier to embryo attachment that must be lifted at the time of implantation. In this study, we investigated the hormonal regulation of human endometrial MUC1 in hormone replacement therapy cycles and in the human blastocyst. We also analyzed the embryonic regulation of MUC1 in human endometrial epithelial cells (EECs) during the apposition and adhesion phases of human implantation using two different in vitro models. Our results indicate that endometrial MUC1 mRNA and immunoreactive protein increase in receptive endometrium compared to nonreceptive endometrium. Human blastocysts express MUC1, as demonstrated by reverse transcription-polymerase chain reaction and immunocytochemistry, localized at the trophectoderm. In vitro, MUC1 was present at the surface of primary cultures of human EEC, and presence of a human blastocyst (i.e., apposition phase) increases EEC MUC1 protein and mRNA compared to control EEC lacking embryos. Interestingly, when human blastocysts were allowed to attach to the EEC monolayer (i.e., adhesion phase), MUC1 was locally removed in a paracrine fashion on EEC at the implantation site. These results demonstrate a coordinated hormonal and embryonic regulation of EEC MUC1. Progesterone combined with estradiol priming induces an up-regulation of MUC1 at the receptive endometrium. During the apposition phase, presence of a human embryo increases EEC MUC1. However, at the adhesion phase, the embryo induces a paracrine cleavage of EEC MUC1 at the implantation site. These findings strongly suggest that MUC1 may act as an endometrial antiadhesive molecule that must be locally removed by the human blastocyst during the adhesion phase.

Adult↗
PubMed2001

Muc1 and glycan expression in the oviduct and endometrium of a New World monkey, Cebus apella.

Cebus apella is a New World monkey that has a menstrual cycle of 18-23 days with implantation at approximately luteal Day 5. The aim of this study was to characterize by lectin- and antibody-labeling the distribution of Muc1 and associated glycans on the endometrial and oviductal epithelium during the luteal phase of the cycle. Endometrial histology showed a thin endometrium, with glands extending deeply into the myometrium. No obvious evidence of secretory differentiation in cells of either the superficial or the basal segments of glands could be obtained using a panel of antibodies and lectins that marked epithelial glycoprotein, and glycosylation changes observed in some other primate endometrial cycles were not observed in this study. Antibodies to human MUC1 were shown to cross-react with C. apella, and Muc1 was localized to the apical epithelial surfaces of both the endometrial and the tubal epithelium, with stronger expression in the latter. Again, no cyclic changes were noted. Antibodies specific to the isoform Muc1/Sec showed strong staining at the apical tubal epithelium, but no reactivity was detectable in the luminal epithelium of the uterus. This observation suggests differences between the two glycocalyces and could help to explain why C. apella embryos do not implant in this location.

Animals↗
PubMed2000

Growth factor-extracellular matrix synergy in the control of trophoblast invasion.

At the periphery of the human placenta, trophoblast attaches to the uterine wall. The tissue interface contains many anchoring sites, with cytotrophoblast columns that form bridges between the overlying extraembryonic (villous) mesenchyme and the maternal decidual stroma beneath. From the periphery of these columns, large numbers of trophoblast cells detach, migrate through the decidua and eventually colonize and transform maternal arteries. In this way the placenta increases and gives priority to the maternal blood supply to the conceptus. We have shown that when early villous tissue is explanted on a collagen gel in serum-free medium, anchoring-site morphogenesis occurs. Thus, in the presence of placental mesenchyme but in the absence of maternal cells, contact with a permissive extracellular matrix (ECM) is necessary and sufficient for cytotrophoblast column development. Proliferation of trophoblast occurs, followed by differentiation into a columnar cell phenotype in which cells remain attached to one another and to the ECM. At this stage, interaction between fibronectin and integrin alpha5beta1 at the cell surface stabilizes the column and the cells remain as a contiguous multilayered sheet. However, the addition of serum-free conditioned medium from first-trimester placental fibroblasts stimulates cytotrophoblast to detach from the distal column and migrate in streams across the ECM. The removal of insulin-like growth factor I (IGF-I) from the fibroblast medium decreases streaming activity, whereas the addition of exogenous IGF-I (10 ng/ml) to serum-free medium produces a streaming phenotype. In contrast, transforming growth factor beta1 (10 ng/ml) maintains the cells in a tight sheet. These results suggest the possibility of a paracrine interaction between villous mesenchyme and cytotrophoblast in anchoring sites to stimulate the infiltration of the maternal ECM by trophoblast. Such a mechanism would be self-limiting because the signal diminishes with distance from the placenta.

Animals↗
PubMed2000

The cell biological basis of human implantation.

Understanding the cellular basis of implantation and placental development depends on a combination of the limited morphological evidence in the human with data from other primates, separate studies of pre-implantation embryos and endometrium and in vitro models. There is increasing evidence of a dialogue between embryo and endometrium that begins prior to implantation and evolves rapidly through the successive epithelial and stromal/vascular phases. This includes paracrine signals passing to endometrial tissue from the embryo, and vice versa. The production and timing of these signals by endometrium, and its ability to respond to signals from the blastocyst, are dependent on steroidal sensitization. A complex cascade of cell adhesion mechanisms and local tissue remodelling are required for the establishment of a stable haemochorial interface.

Cell Division↗
PubMed2000

Mosaic characteristics of human endometrial epithelium in vitro: analysis of secretory markers and cell surface ultrastructure.

Specific terminal carbohydrate structures and mucin-associated glycans increase in expression within the human endometrial epithelium during the secretory phase of the menstrual cycle but exhibit wide intercellular variation. We postulated that variation in glycosylation between cells would produce differences in the glycocalyx and result in complex mixtures of cells bearing different combinations of glycans. MUC-1 mucin, keratan sulphate and fucosylated lactosaminoglycans were examined in epithelial gland fragment cultures with antibodies (HMFG1, 5D4) and a lectin (Dolichos biflorus agglutinin). The glycocalyx was examined by transmission and high resolution scanning electron microscopy. The data were related to patterns of expression seen in vivo. The MUC-1 mucin was expressed relatively uniformly in culture, but heterogeneity was evident in mucin sialylation within the epithelial cell population. Double labelling of gland explant cultures for combinations of fucosylated lactosaminoglycans, keratan sulphate and MUC-1 demonstrated cells expressing all combinations of these markers. Ultrastructural examination confirmed remarkable intercellular variation in the glycocalyx. Though the human endometrial epithelium is relatively morphologically homogeneous, these observations reveal complex variations of cell surface glycosylation between neighbouring cells and suggest that secretory function might vary in a similar fashion.

Adult↗