Browsing by Author "Millar, Robert"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Open Access
    The differential expression of Kiss1, MMP9 and angiogenic regulators across the feto-maternal interface of healthy human pregnancies: implications for trophoblast invasion and vessel development
    (Public Library of Science, 2013) Matjila, Mushi; Millar, Robert; van der Spuy, Zephne; Katz, Arieh
    Genes involved in invasion of trophoblast cells and angiogenesis are crucial in determining pregnancy outcome. We therefore studied expression profiles of these genes in both fetal and maternal tissues to enhance our understanding of feto-maternal dialogue. We investigated the expression of genes involved in trophoblast invasion, namely Kiss1, Kiss1 Receptor (Kiss1R) and MMP9 as well as the expression of angiogenic ligands Vascular Endothelial Growth Factor-A ( VEGF-A) and Prokineticin-1 ( PROK1 ) and their respective receptors (VEGFR1, VEGFR2 and PROK1R ) across the feto-maternal interface of healthy human pregnancies. The placenta, placental bed and decidua parietalis were sampled at elective caesarean delivery. Real-time RT-PCR was used to investigate transcription, while immunohistochemistry and western blot analyses were utilized to study protein expression. We found that the expression of Kiss1 (p<0.001), Kiss1R (p<0.05) and MMP9 (p<0.01) were higher in the placenta compared to the placental bed and decidua parietalis. In contrast, the expression of VEGF-A was highest in the placental bed ( p<0.001 ). While VEGFR1 expression was highest in the placenta (p<0.01), the expression of VEGFR2 was highest in the placental bed (p<0.001). Lastly, both PROK1 (p<0.001) and its receptor PROK1R (p<0.001) had highest expression in the placenta. Genes associated with trophoblast invasion were highly expressed in the placenta which could suggest that the influence on invasion capacity may largely be exercised at the fetal level. Furthermore, our findings on angiogenic gene expression profiles suggest that angiogenesis may be regulated by two distinct pathways with the PROK1/PROK1R system specifically mediating angiogenesis in the fetus and VEGFA/VEGFR2 ligand-receptor pair predominantly mediating maternal angiogenesis.
  • Thumbnail Image
    Item
    Open Access
    Interactions of GPR54 and GPR147 receptors with RF-amide ligands
    (2014) Hendrikse, Megan; Katz, Arieh; Millar, Robert
    G protein-coupled receptors play a key role in cellular signaling by transducing extracellular signals via G proteins to elicit intracellular responses. Studies have provided evidence supporting the role of the GPCR GPR54 and its cognate peptide ligand, kisspeptin (an RFamide peptide), in the regulation of reproduction. Kisspeptin and GPR54 play a critical role in the control of the hypothalamic-pituitary-gonadal axis by regulating gonadotropin-releasing hormone secretion. Despite the physiological importance of GPR54/kisspeptin signalling, the GRP54 residues important for receptor activation and signalling have not been extensively investigated. Another hypothalamic peptide, gonadotropin inhibiting hormone (also known as RFamide-related peptide), which interacts with the GPCR GPR147, has been found to inhibit GnRH-induced gonadotropin release and is therefore also of importance in control of the HPG axis. As many of the RFamide and RFamide-related receptors and ligands can be promiscuous, there is the potential for crosstalk between the GPR54/kisspeptin and GRP147/RFRP systems (or other RFamides) which may be of importance in the regulation of reproduction. GPR54 chimeras and point mutants were constructed in order to investigate the residues important for kisspeptin binding and receptor activation. The data obtained indicate that the acidic residues within the extracellular loops of GPR54 contribute to cell surface receptor expression and play a role in receptor signalling. In order to investigate the interactions of kisspeptin/RFRP peptides at GPR147 and GPR54, binding and activation of these receptors was studied with a range of ligands and their analogs. In addition to RFRP and its analogs, kisspeptin and several kisspeptin analogs were found to act as agonists at GRP147. In contrast, of all the ligands tested, only kisspeptin was able to bind to GPR54 with high affinity and elicit a response, thus indicating that GPR54 has high specificity for kisspeptin in contrast to the more promiscuous GPR147. These data demonstrate the therapeutic potential of kisspeptin analogs, for the inhibition of gonadotropin secretion and treatment of sex steroid hormone disease. In addition, these data have identified ligand and receptor residues important for binding and activation of GRP54/GRP147 which may aid development of new analogs targeting these receptors and highlighted the importance of testing these analogs for receptor specificity.
  • Thumbnail Image
    Item
    Open Access
    The role of extracellular loop three of the human gonadotropin releasing hormone receptor in ligand selectivity
    (2001) Fromme, Bernhard Johannes; Millar, Robert
    The hypothalamic neuropeptide, gonadotropin releasing hormone (GnRH) perferentially inteacts with GnRH type I receptors on the gonadotropes in the anterior pituitary. Activation of the GnRH receptor is required for the biosynthesis and release of luteinizing hormone (LH) and follicle stimulating hormone (FSH), which regulate reproductive function. Multiple forms of GnRH are present in most vertebrate species and are thought to have physiological functions in addition to regulating pituitary hormone release. The mammalian type I GnRH receptor is proposed to discriminate between endogenous forms of GnRH (King and Millar, 1995). In this thesis the mechanism of the GnRH selectivity by a mammalian type I GnRH receptor is examined at molecular level and previous hypotheses are re-evaluated.
  • Thumbnail Image
    Item
    Open Access
    The role of kisspeptin and its cognate receptor GPR54 in normal and abnormal placentation
    (2015) Matjila, Mushi Johannes; van der Spuy, Zephne Margaret; Katz, Arieh; Millar, Robert
    Poor invasion of trophoblast cells in early pregnancy has been associated with preeclampsia and intrauterine growth restriction as well as other adverse pregnancy outcomes such as miscarriage, preterm birth and intrauterine death. Hypertensive disorders of pregnancy, including pre-eclampsia are one of the leading causes of maternal mortality in South Africa (Third report on Confidential Enquiries into Maternal Deaths in South Africa (2002-2004)) and the rest of the world. The currently accepted mechanism underlying the development of preeclampsia implicates poor trophoblast invasion and inadequate transformation of the maternal spiral arteries. Despite extensive research in this area, the control of trophoblast invasion and early placental development remains poorly understood. A whole host of factors such as oxygen tension, activation of matrix metalloproteinases (MMPs), angiogenic factors (VEGF-A) and immunological factors such as TNF alpha, interleukins and TGFβ have been shown to be involved in the control of trophoblast invasion. Our knowledge of the molecular details of pregnancy is unfortunately limited to in-vitro experiments and animal studies. Recently kisspeptins and their cognate receptor GPR-54 originally involved in tumour metastasis suppression and regulation of puberty, have been implicated in the inhibition of trophoblast invasion. Expression levels of kisspeptin and its receptor in trophoblast cells are highest in the first trimester, when control of trophoblast invasion is critical, and lower towards term.