Implantation and growth of the placenta requires extensive angiogenesis to establish the vascular structures involved in exchange. Failure to establish adequate blood supply to the fetus may have serious clinical consequences such as intrauterine growth retardation. Vascular endothelial cell growth factor (VEGF) is a recently identified growth factor with significant angiogenic properties. We have demonstrated the presence of four species of mRNA encoding VEGF in both first trimester and term placenta. In situ hybridization was used to localize the sites of expression of VEGF mRNA in these tissues. VEGF expression was seen in villous trophoblast in the first trimester and in extravillous trophoblast at term, and in both fetal macrophages within the villi and maternal macrophages in the decidua. Glandular epithelium in maternal decidua also expressed VEGF mRNA. The strongest site of expression was in maternal macrophages adjacent to Nitabuch's stria, a zone of necrosis at the site of implantation. This complex pattern of expression suggests that VEGF is involved in angiogenesis on both maternal and fetal sides of the placenta and that macrophages are the primary source of VEGF. However, VEGF may also play a role in term placenta, when extensive angiogenesis has diminished, possibly regulating vascular permeability.
A. M. Sharkey, D. S. Charnock-Jones, C. A. Boocock, K. D. Brown and S. K. Smith
J. C. Cooper, A. M. Sharkey, J. McLaren, D. S. Charnock-Jones and S. K. Smith
Vascular endothelial growth factor is a secreted angiogenic growth factor the mRNA of which is present in the placenta. The mRNA encoding the vascular endothelial growth factor receptor, flt, has also been demonstrated in placenta, with trophoblast appearing to be a novel site of flt expression. We investigated the expression of both vascular endothelial growth factor and flt-like immunoreactivity in first trimester and term placentae. In the first trimester, vascular endothelial growth factor immunoreactivity was localized to placental macrophages (Hofbauer cells), and in decidua, to glandular epithelium and maternal macrophages. In the term placenta, vascular endothelial growth factor immunoreactivity was present in extravillous trophoblast and in extracellular material. Flt immunoreactivity was demonstrated on extravillous trophoblast in first trimester and term, and on Hofbauer cells within placental villi. This complex pattern of both vascular endothelial growth factor and flt-like immunoreactivity suggests that vascular endothelial growth factor may be involved not only in the regulation of placental angiogenesis, but also in trophoblast invasion.
C L Tower, S Lui, N R Charlesworth, S D Smith, J D Aplin and R L Jones
Angiotensin II (Ang II) is locally generated in the placenta and regulates syncytial transport, vascular contractility and trophoblast invasion. It acts through two receptor subtypes, AGTR1 and AGTR2 (AT1 and AT2), which typically mediate antagonising actions. The objectives of this study are to characterise the cellular distribution of AGTR1 and AGTR2 at the maternal–fetal interface and explore the effects on cytotrophoblast turnover. Low levels of AGTR2 mRNA were detected in first trimester placental homogenates using real-time PCR. Immunohistochemistry using polyclonal antibodies against AGTR1 and AGTR2 detected the receptors in first trimester placenta, decidua basalis and villous tip outgrowths in culture. Serial staining with cytokeratin-7 was used to identify extravillous trophoblasts (EVTs). AGTR1 was found in the syncytiotrophoblast microvillous membrane, in a subpopulation of villous cytotrophoblasts, and in Hofbauer cells. AGTR1 was strongly upregulated in cytotrophoblasts in cell columns and villous tip outgrowths, but was absent in interstitial and endovascular EVTs within the decidua. AGTR2 immunostaining was present in Hofbauer cells and villous cytotrophoblasts, but was absent from syncytiotrophoblast. Faint staining was detected in cell column cytotrophoblasts and villous outgrowths, but not in EVTs within the decidua. Both receptors were detected in placental homogenates by western blotting. Ang II significantly increased proliferation of cytotrophoblasts in both villous explants and villous tip outgrowths, but did not affect apoptosis. Blockade of AGTR1 and AGTR2 together abrogated this effect. This study shows specific expression patterns for AGTR1 and AGTR2 in distinct trophoblast populations at the maternal–fetal interface and suggests that Ang II plays a role in placental development and generation of EVTs.
C. J. P. Jones, S. J. Kimber, I. Illingworth and J. D. Aplin
Biotinylated lectins from Sambucus nigra (SNA) and Maackia amurensis (MAA), which bind to α2,6-linked and α2,3-linked sialyl residues, respectively, were used as probes to study glycan terminal modifications associated with decidualization in the uterine stroma of pregnant rats and mice. Binding of lectins from Erythrina cristagalli (ECA), Phaseolus vulgaris (leukoagglutinin, L-PHA), Triticum vulgaris (WGA) and Bandeiraea simplicifolia (BSA-1B4) was also examined. Tissues from rats between day 5 and day 8 of gestation and mice between day 5 and day 7 of gestation were fixed in Bouin's solution and embedded in wax prior to lectin histochemistry. On day 7 in rats and day 6 in mice, there was a marked reduction in the binding of SNA in the subluminal decidua surrounding the implantation site. In rats, MAA binding to enlarged decidual cells around the implantation chamber was increased markedly, but there was no change in mice. In both species there was de novo binding of ECA in the SNA-negative area, suggesting that the loss of α2,6-linked sialyl residues unmasks terminal N-acetyl lactosamine. These findings are consistent with previous evidence of a close structural and functional similarity between the artificially induced deciduoma and true decidua of rats and show identical changes to the glycosylation patterns previously found in differentiating rat deciduoma. In both species, therefore, decidua exhibits regionally specific terminal glycosylation. However, the species-specific expression of α2,3-linked sialyl residues suggests distinct patterns of steroidally modulated sialyl transferase expression.
C. J. P. Jones, J. D. Aplin, J. Mulholland and S. R. Glasser
Lectin histochemistry was used to demonstrate changes in the surface glycan distribution of uterine stromal cells as they differentiate to form decidual cells. Decidualization was induced in hormone-treated, ovariectomized rat uteri by needle scratch. Uterine tissue from days 2 to 8 of deciduoma development was examined with a panel of lectins specific for terminal nonreducing structures in N- and O-linked classes of glycoprotein glycan, including α2,3- and α2,6-linked sialic acid residues. Immunostaining for desmin was used to identify decidual cells. An increase in N-linked glycans associated with the cell surface and recognized by lectins from Phaseolus vulgaris (leukoagglutinin) (1-PHA), Pisum sativum (PSA) and Triticum vulgaris (WGA) was found during the early growth of decidual cells. As decidualization progressed regionally from the antimesometrial to mesometrial uterus, an increase in α2,3-linked sialic acid residues was followed by a loss of the α2,6-linked form. The results suggest that as stromal cells differentiate, glycoprotein biosynthesis and glycosyl transferase activity are altered. These changes in patterns of glycosylation may give rise to altered decidual cell–matrix and cell–cell interactions during differentiation and play a role in the modulation of decidual cell interactions with trophoblast during early placentation.
M. J. K. Harper, M. A. Jones, C. J. Norris and D. S. Woodard
Summary. Day-6 rabbit blastocysts were recovered from superovulated donor animals, washed in ice-cold Krebs–Ringer–bicarbonate (KRB) buffer, pooled and randomly allocated to polypropylene incubation tubes, usually 10 blastocysts in 1 ml KRB. The blastocysts were ruptured with a dissecting needle and incubated at 37°C for periods of 1–3 h with 10 μCi [3H]arachidonic acid/tube. A control tube without blastocysts was run in each experiment. At the end of the incubation, the samples were acidified, extracted with ethyl acetate, dried down and resuspended in h.p.l.c. column solvent. The radioactivity from the control tube eluting from the h.p.l.c., using a solvent system for prostaglandins (PGs), was subtracted from each experimental run in the same experiment. The remaining radioactivity constituted 0·14% of the original [3H]arachidonic acid added to each incubation tube. This was considered to have been the result of conversion of the radiolabelled arachidonic acid to prostanoids. In the absence of 10 mm-EDTA no conversion occurred, whereas in its presence peaks of radioactivity co-eluting with [3H]PGF-2α and [3H]PGE-2 were seen. A third peak that eluted was either 15-keto metabolites of these PGs or PGD-2. These 3 peaks were always significantly above background, and usually did not differ from each other. No differences in amount of conversion could be related to incubation time. Addition of indomethacin (100 μg/ml) or radioinert arachidonic acid (10 μg/ml) inhibited production of [3H]PG, even in the presence of EDTA. Removal of calcium from the incubation medium was per se without effect. Addition of atropine (0·15 mm) or carbachol (0·15 mm) in the presence or absence of EDTA did not change the pattern of conversion of [3H]arachidonic acid to [3H]PGs. These experiments demonstrate that rabbit blastocysts have the capacity for de-novo synthesis of PGs from exogenous substrate, when utilization of endogenous substrate is inhibited. The extent of conversion observed may not be a true reflection of the capacity for conversion of endogenous substrate.
Keywords: rabbit; blastocysts; prostaglandins
S J Calvert, M S Longtine, S Cotter, C J P Jones, C P Sibley, J D Aplin, D M Nelson and A E P Heazell
Syncytial nuclear aggregates (SNAs), clusters of nuclei in the syncytiotrophoblast of the human placenta, are increased as gestation advances and in pregnancy pathologies. The origins of increased SNAs are unclear; however, a better appreciation of the mechanism may give insight into placental ageing and factors underpinning dysfunction. We developed three models to investigate whether SNA formation results from a dynamic process of nuclear movement and to generate alternative hypotheses. SNA count and size were measured in placental explants cultured over 16 days and particles released into culture medium were quantified. Primary trophoblasts were cultured for 6 days. Explants and trophoblasts were cultured with and without cytoskeletal inhibitors. An in silico model was developed to examine the effects of modulating nuclear behaviour on clustering. In explants, neither median SNA number (108 SNA/mm2 villous area) nor size (283 μm2) changed over time. Subcellular particles from conditioned culture medium showed a wide range of sizes that overlapped with those of SNAs. Nuclei in primary trophoblasts did not change position relative to other nuclei; apparent movement was associated with positional changes of the syncytial cell membrane. In both models, SNAs and nuclear clusters were stable despite pharmacological disruption of cytoskeletal activity. In silico, increased nuclear movement, adhesiveness and sites of cytotrophoblast fusion were related to nuclear clustering. The prominence of SNAs in pregnancy disorders may not result from an active process involving cytoskeleton-mediated rearrangement of syncytial nuclei. Further insights into the mechanism(s) of SNA formation will aid understanding of their increased presence in pregnancy pathologies.
R. E. Zimmerman, R. S. Nevin, D. J. Allen, C. D. Jones, M. E. Goettel and P. J. Burck
Summary. Acrosin and acrosomal hyaluronidase were inhibited by tetradecyl sodium sulphate (TDSS) in vitro at concentrations of < 10−4 M. TDSS prevented the removal in vitro of the cumulus oophorus by testicular hyaluronidase and the zona pellucida by acrosin. TDSS had a contraceptive effect in rabbits when administered intravaginally before coitus or released at levels of 1–3 μg/day from intrauterine silicone rubber devices.
H. J. Stewart, D. S. C. Jones, J. C. Pascall, R. M. Popkin and A. P. F. Flint
Page Introduction 2 Structures of genes and mRNA molecules 2 Structure of genes 2 Sequences of promoters and enhancers 3 Signals for terminating RNA synthesis 5 Structure of mRNA molecules 5 Structure of genes coding for reproductive polypeptide hormones and hypothalamic releasing factors 7 Structure of gonadotrophin genes 7 Structure of placental lactogen, prolactin and growth hormone genes 10 Structure of the LHRH gene 12 Inhibin, activin and Müllerian duct-inhibiting hormone 13 Localization of gene products by in-situ hybridization 16 Pre- and post-translational processing 18 Pretranslational processing: differential splicing of RNA 18 Post-translational processing 18 Steroidogenic enzymes and steroid sulphatase 20 Steroid hormone receptors 22 Steroid receptor primary structure: amino acid sequences derived from cloned DNA 23 Steroid hormone receptors and oncogenesis 26 Mechanism of binding of receptors to DNA—the finger hypothesis 27 DNA sequences recognized by steroid receptors in eukaryotic cells 28 Fertilization and early development 30 Expression of