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Fetal programming of adult disease: Causes and consequences of metabolic dysregulation in an ovine model of PCOS Katarzyna Siemienowicz The University of Edinburgh Thesis submitted for the fulfilment of the degree of Doctor of Philosophy August 2017 Contents Acknowledgements ................................................................................................................ ii Abstract .................................................................................................................................. iii Lay abstract ............................................................................................................................ v Presentations relating to this thesis .................................................................................... vii Abbreviations ...................................................................................................................... viii Chapter 1 Literature Review ............................................................................................. 1 1.1 Female reproductive function ................................................................................. 2 1.1.1 Mammalian reproductive development .............................................................. 2 1.1.2 The hypothalamic-pituitary-ovarian axis ........................................................... 3 1.1.3 Folliculogenesis.................................................................................................. 4 1.1.4 Female puberty ................................................................................................... 6 1.1.5 Human ovarian cycle .......................................................................................... 7 1.1.5.1 The follicular phase ......................................................................................... 7 1.1.5.2 The luteal phase .............................................................................................. 7 1.1.6 Ovarian steroidogenesis ..................................................................................... 8 1.1.7 Adrenal steroidogenesis ..................................................................................... 9 1.1.8 Ovarian and adrenal androgens in adult females ............................................. 10 1.1.9 Ovarian and adrenal androgens in adolescent girls .......................................... 12 1.1.10 Reproduction in sheep ................................................................................. 12 1.1.11 Regulation of pubertal transition in sheep ................................................... 13 1.2 Role of adipose tissue in metabolism ................................................................... 14 1.2.1 Adipocyte development and growth ................................................................ 15 1.2.2 Lipolysis and triglyceride formation ................................................................ 16 1.2.3 WAT as an endocrine organ ............................................................................. 16 1.2.3.1 Leptin ............................................................................................................ 17 1.3.2.2 Adiponectin ................................................................................................... 18 1.2.4 Adipose tissue depot differences between SAT and VAT ............................... 19 1.2.5 Central obesity ................................................................................................. 20 1.2.6 Obesity and WAT ............................................................................................ 20 1.2.7 Role of brown adipose tissue in energy homeostasis ....................................... 22 1.2.8 Adipose tissue and sexual dimorphism ............................................................ 23 1.3 Clinical features and diagnostic criteria of PCOS ................................................ 24 1.3.1 Clinical features and diagnostic criteria of PCOS in adolescent girls .............. 26 1.3.2 Prevalence of PCOS ......................................................................................... 26 1.3.3 Metabolic characteristics of adult women with PCOS ..................................... 27 1.3.3.1 Insulin resistance ........................................................................................... 27 1.3.3.2 Type 2 diabetes mellitus ................................................................................ 27 1.3.3.3 Obesity .......................................................................................................... 28 1.3.3.4 Adipose tissue dysfunction ............................................................................ 29 1.3.3.5 Metabolic syndrome ...................................................................................... 30 1.3.3.6 Non-alcoholic fatty liver disease ................................................................... 31 1.3.3.7 Dyslipidaemia ................................................................................................ 31 1.3.3.8 Cardiovascular disease .................................................................................. 32 1.3.4 Metabolic characteristics of adolescent girls with PCOS ................................. 32 1.3.4.1 Insulin resistance ........................................................................................... 32 1.3.4.2 Obesity .......................................................................................................... 33 1.3.4.3 Metabolic syndrome ...................................................................................... 34 1.3.5 Pathophysiology of PCOS ................................................................................ 35 1.3.6 Management of PCOS ...................................................................................... 38 1.3.6.1 Insulin sensitisers .......................................................................................... 38 1.3.6.2 Oral contraceptives ........................................................................................ 39 1.4 Aetiology of PCOS ............................................................................................... 40 1.4.1 Heritability of PCOS ........................................................................................ 40 1.4.2 Genetics of PCOS ............................................................................................. 40 1.4.3 Prenatal programming of PCOS ....................................................................... 42 1.5 Animal models of PCOS ....................................................................................... 45 1.5.1 Non-human primate model ............................................................................... 45 1.5.2 Ovine model ..................................................................................................... 45 1.6 The objectives of this thesis .................................................................................. 47 Chapter 2 Materials and Methods .................................................................................. 49 2.1 Introduction ........................................................................................................... 50 2.2 Animal husbandry ................................................................................................. 50 2.2.1 Mating and pregnant ewe husbandry ................................................................ 50 2.2.2 Pregnant ewe treatment .................................................................................... 51 2.2.2.1 Maternal Injections ........................................................................................ 51 2.2.2.2 Fetal Injections .............................................................................................. 51 2.2.3 Non-pregnant treatment .................................................................................... 52 2.2.4 Husbandry of offspring ..................................................................................... 52 2.2.5 Animal sacrifice and specimen collection ........................................................ 53 2.2.5.1 Animal sacrifice ............................................................................................ 53 2.2.5.2 Tissue and plasma collection ........................................................................ 53 2.3 Histology .............................................................................................................. 55 2.3.1 Tissue Processing ............................................................................................