Regulation Of Myometrial Gene Expression By Oestrogen And Progesterone: The Use Of A Cell Culture Model To Mimic The In Vivo Conditions Of Pregnant Human Uterus

i Acknowledgement ii Abbreviations iii List of tables vi List of figures viii Chapter 1 Introduction 1.1 Pregnancy 2 1.2 Female reproductive system 2 1.3 Fertilisation 4 1.3. 1 Blastocyst development 5 1.3. 2 Embryo development 6 1.3. 3 Foetal and placental development 6 1.4 Gestation length 7 1.4. 1 Preterm (premature) delivery 8 1.4. 2 Post-term (postmature) delivery 9 1.5 Utero-placental tissues in pregnancy 9 1.5.1 Amnion 9 1.5.2 Chorion 10 1.5.3 Placenta 10 1.5. 4 Cervix 11 1.5. 5 Decidua 11 1.5. 6 Myometrium 12 1.5. 6.1 Smooth muscle and its structure 12 1.5. 6.2 Uterine smooth muscle contraction and relaxation 13 1.6 Hormones and peptides in pregnancy and labour 16 1.6.1 Oxytocin (OT) 17 1.6.2 Prostaglandins (PG) 18 1.6.3 Corticotropin-releasing hormone (CRH) 19 1.6. 4 Relaxin (RLX) 21 1.6. 5 Oestrogen 22 1.6. 5.1 Oestrogen synthesis during pregnancy 22 1.6. 5.2 Oestrogen levels during parturition 25 1.6. 5.3 Role of oestrogen in human parturition 26 1.6. 6 Progesterone 29 1.6. 6.1 Progesterone synthesis during human pregnancy 30 1.6. 6.2 Changes in the progesterone levels at parturition 30 1.6. 6.3 Role of progesterone in human parturition 31 1.7 Steroid hormone receptor (SHR) dynamics during pregnancy and labour 32 1.7. 1 Oestrogen receptors (ER) 35 1.7. 2 Progesterone receptors (PR) 35 1.8 Parturition 37 1.8. 1 Phases of parturition 38 1.8. 2 Parturition in humans and other animals 38 1.9 Functional genomic studies of pregnant and labouring human myometrium 41 1.10 Uterine smooth muscle cell culture system 47 1.11 Aims of Study 49 Chapter 2 – Materials and Methods 2.1 Materials 51 2.2 Methods 51 2.2.1 Culture of human uterine smooth muscle cells 51 2.2.2 Cryopreservation 52 2.2. 3 Resuscitation 52 2.2. 4 Preconditioning the cells for serum starvation 52 2.2. 5 Serum starvation 52 2.2. 6 17β-Estradiol (E2) treatment 53 2.2. 7 Progesterone (P4) and Mifepristone (RU486) treatment 55 2.2. 8 Cell lysate preparation 57 2.2. 9 Protein assay 57 2.2.10 Sodium Dodecyl Sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) 57 2.2.11 Western blotting 58 2.2.12 Isolation of RNA 60 2.2.13 Quantitation of RNA 60 2.2.14 Quality control of RNA 61 2.2.15 cDNA synthesis 61 2.2.16 Polymerase chain reaction (PCR) 62 2.2.17 Agarose gel electrophoresis 63 2.2.18 Quantitative real-time PCR 63 2.2.19 TaqMan small RNA assay 65 2.2.20 Microarray analysis 66 2.2.20.1 First strand synthesis 67 2.2.20.2 Second strand cDNA synthesis and purification 68 2.2.20.3 In vitro transcription to synthesise amino allyl-modified aRNA 68 2.2.20.4 Amino allyl-modified aRNA purification 69 2.2.20.5 Amino allyl-modified aRNA quality control 69 2.2.20.6 Amino allyl-modified aRNA labelling 69 2.2.20.7 Labelled amino allyl-modified aRNA purification 69 2.2.20.8 Labelled amino allyl-modified aRNA fragmentation 70 2.2.20.9 Pre-hybridisation, hybridisation and washing 70 2.2.20.10 Scanning 72 2.2.20.11 Image analysis 72 2.2.20.12 GeneSpring GX analysis 74 2.2.21 Gene Ontology analysis 75 2.2.22 Pathway analysis 76 2.2.23 Statistical analysis 77 Chapter 3: Characterisation and Optimisation of Human Uterine Smooth Muscle Cell Culture System